IL-15-based fusions to IL-7 and IL-21

ABSTRACT

The invention features multi-specific fusion protein complexes with one domain comprising IL-15 or a functional variant and a binding domain specific to IL-7 or IL-21.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application62/551,218 filed on Aug. 28, 2017, the entire contents of which areincorporated herein by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 18, 2018, isnamed 048277-533001US_SL.txt and is 9.51 bytes in size.

FIELD OF THE INVENTION

This invention relates generally to the field of multimeric fusionmolecules.

BACKGROUND OF THE INVENTION

Prior to the invention described herein, there was a pressing need todevelop new strategies to target various effector molecules to a diseasesite to provide therapeutic benefit without the side effects associatedwith non-specific immune activity.

SUMMARY OF THE INVENTION

The invention is based, at least in part, on the surprising discoverythat multi-specific interleukin-15 (IL-15)-based protein complexesenhance the stimulation of immune cells and promote their activityagainst disease cells, thereby resulting in reduction or prevention ofdisease. These IL-15-based protein complexes also are capable of bindingto disease and target antigens. Provided herein are multi-specificIL-15-based protein complexes comprising IL-7 and IL-21 binding domains(FIGS. 1A-1C). Specifically, described herein are protein complexescomprising an IL-15N72D:IL-15RαSu-Ig Fc scaffold fused to IL-7 and IL-21binding domains.

As described in detail below, when characterized using human immunecells, these complexes exhibit binding and biological activity of eachof the IL-15, IL-7 and IL-21 cytokines. Additionally, these complexesinduce proliferation and activation of both T cells and natural killer(NK) cells with enhanced production of IFN-γ. Surprisingly, thesecomplexes were capable of inducing immune responses to a greater degreethan was observed by the individual cytokines alone or in combination.

As such, the complex as a single molecule binds to and signals viamultiple cytokine receptors on NK and T cells to provide the responsespreviously observed only with a combination of multiple cytokines.Additionally, these complexes comprise the Fc region of Ig molecules,which can form a dimer to provide a soluble multi-polypeptide complex,bind Protein A for the purpose of purification and interact with Fcγreceptors on NK cells and macrophages, thereby providing advantages tothe complex that are not present in the combination of individualcytokines. Mammalian cell expression-based methods for making thesecomplexes suitable for large scale production of clinical grade materialare described herein. Additional methods for making and using NK and Tcells which proliferate following induction by the protein complex ofthe invention are also provided.

Accordingly, provided is an isolated soluble fusion protein complexcomprising at least two soluble proteins. For example, the first proteincomprises an IL-15 polypeptide, e.g., a variant IL-15 polypeptidecomprising an N72D mutation (IL-15N72D). The second protein comprises asoluble IL-15 receptor alpha sushi-binding domain (IL-15RαSu) fused toan immunoglobulin Fc domain (IL-15RαSu/Fc). A third component of theisolated soluble fusion protein complex comprises a binding domain ofIL-7, wherein the IL-7 binding domain is fused to the either theIL-15N72D or the IL-15RαSu/Fc protein. A fourth component of theisolated soluble fusion protein complex comprises a binding domain ofIL-21, wherein the IL-21 binding domain is fused to the either theIL-15N72D or the IL-15RαSu/Fc protein. In some cases, the IL-7 and/orIL-21 binding domains are fused to both the IL-15N72D and IL-15RαSu/Fcproteins. In other cases, either the IL-7 or IL-21 binding domain isfused to the IL-15N72D or the IL-15RαSu/Fc proteins and another bindingdomain is fused to the other protein. An exemplary fusion proteincomplex comprises an IL-7 polypeptide covalently linked to an IL-15N72Dand an IL-21 polypeptide covalently linked to an IL-15RαSu/Fc fusionprotein (FIGS. 1A, 1B).

Exemplary first proteins comprise the amino acid sequences set forth inSEQ ID NO: 2 and SEQ ID NO: 4. Exemplary nucleic acid sequences encodingthe first protein comprise the sequences set forth in SEQ ID NO: 1 andSEQ ID NO: 3. In one aspect, the nucleic acid sequence(s) furthercomprises a promoter, translation initiation signal, and leader sequenceoperably linked to the sequence encoding the fusion protein. Alsoprovided are DNA vectors comprising the nucleic acid sequences describedherein. For example, the nucleic acid sequence is in a vector forreplication, expression, or both.

Also provided is a soluble fusion protein complex comprising a firstsoluble fusion protein complex covalently linked to a second solublefusion protein complex. For example, the soluble fusion proteincomplexes of the invention are multimerized, e.g., dimerized,trimerized, or otherwise multimerized (e.g., 4 complexes, 5 complexes,etc.). For example, the multimers are homomultimers or heteromultimers.The soluble fusion protein complexes are joined by covalent bonds, e.g.,disulfide bonds, chemical cross-linking agents. In some cases, onesoluble fusion protein is covalently linked to another soluble fusionprotein by a disulfide bond linking the Fc domain of the first solubleprotein to the Fc domain of the second soluble protein.

The Fc domain or functional fragment thereof includes an Fc domainselected from the group consisting of IgG Fc domain, human IgG1 Fcdomain, human IgG2 Fc domain, human IgG3 Fc domain, human IgG4 Fcdomain, IgA Fc domain, IgD Fc domain, IgE Fc domain, and IgM Fc domain;mouse IgG2A domain, or any combination thereof. Optionally, the Fcdomain includes an amino acid change that results in an Fc domain withaltered complement or Fc receptor binding properties or altereddimerization or glycosylation profiles. Amino acid changes to produce anFc domain with altered complement or Fc receptor binding properties oraltered dimerization or glycosylation profiles are known in the art. Forexample, a substitution of leucine residues at positions 234 and 235 ofthe IgG1 CH2 (numbering based on antibody consensus sequence) (i.e., . .. P E L L G G . . . ) with alanine residues (i.e., . . . P E A A G G . .. ) results in a loss of Fc gamma receptor binding, whereas thesubstitution of the lysine residue at position 322 of the IgG1 CH2(numbering based on antibody consensus sequence) (i.e., . . . K C K S L. . . ) with an alanine residue (i.e., . . . K C A S L . . . ) resultsin a loss of complement activation. In some examples, such mutations arecombined.

In some aspects, the IL-7 or IL-21 binding domain is covalently linkedto an IL-15 polypeptide (or functional fragment thereof) by apolypeptide linker sequence. Similarly, the IL-7 or IL-21 binding domainis covalently linked to an IL-15Rα polypeptide (or functional fragmentthereof) by a polypeptide linker sequence. Optionally, the IL-15Rαpolypeptide (or functional fragment thereof) is covalently linked to theFc domain (or functional fragment thereof) by a polypeptide linkersequence. Each polypeptide linker sequence can be selectedindependently. Optionally, the polypeptide linker sequences are thesame. Alternatively, they are different.

Optionally, the soluble fusion protein complexes of the invention areprovided wherein at least one of the soluble fusion proteins compriseone or more binding domain or detectable labels. Such binding domainsmay comprise antibodies, soluble T cell receptors, ligands, solublereceptor domains or functional fragments thereof. IL-15-based fusionprotein complexes comprising such binding domains have been previouslydescribed in U.S. Pat. No. 8,492,118, incorporated herein by reference.Detectable labels include, but are not limited to, biotin, streptavidin,an enzyme or catalytically active fragment thereof, a radionuclide, ananoparticle, a paramagnetic metal ion, or a fluorescent,phosphorescent, or chemiluminescent molecule, or any combinationthereof.

The invention provides methods for making the soluble fusion proteincomplexes of the invention. The method includes the steps of: a)introducing into a first host cell a DNA vector with appropriate controlsequences encoding the first protein, b) culturing the first host cellin media under conditions sufficient to express the first protein in thecell or the media; c) purifying the first protein from the host cells ormedia, d) introducing into a second host cell a DNA vector withappropriate control sequences encoding the second protein, e) culturingthe second host cell in media under conditions sufficient to express thesecond protein in the cell or the media; and f) purifying the secondprotein from the host cells or media, and g) mixing the first and secondproteins under conditions sufficient to allow binding between IL-15domain of a first protein and the soluble IL-15Rα domain of a secondprotein to form the soluble fusion protein complex.

In some cases, the method further includes mixing the first and secondprotein under conditions sufficient to allow formation of a disulfidebond between the polypeptides expressed from the expression vectors.

Alternatively, methods for making soluble fusion protein complexes ofthe invention are carried out by a) introducing into a host cell a DNAvector with appropriate control sequences encoding the first protein anda DNA vector with appropriate control sequences encoding the secondprotein, b) culturing the host cell in media under conditions sufficientto express the proteins in the cell or the media and allow associationbetween IL-15 domain of a first protein and the soluble IL-15Rα domainof a second protein to form the soluble fusion protein complex; and c)purifying the soluble fusion protein complex from the host cells ormedia.

In one aspect, the method further includes mixing the first and secondprotein under conditions sufficient to allow formation of a disulfidebond between the polypeptides expressed from the expression vectors.

Also provided are methods for making soluble fusion protein complexescomprising a) introducing into a host cell a DNA vector with appropriatecontrol sequences encoding the first and second proteins, b) culturingthe host cell in media under conditions sufficient to express theproteins in the cell or the media and allow association between IL-15domain of a first protein and the soluble IL-15Rα domain of a secondprotein to form the soluble fusion protein complex, and to allowformation of a disulfide bond between the polypeptides; and c) purifyingthe soluble fusion protein complex from the host cells or media.

Optionally, the method further includes mixing the first and secondprotein under conditions sufficient to allow formation of a disulfidebond between the polypeptides expressed from the expression vectors.

In some cases, the method further includes purification of the complexby Protein A affinity chromatography, size exclusion chromatography, ionexchange chromatography and/or other standard methods (including viralinactivation and/or filtration) sufficient to generate a sufficientlypure protein complex suitable for use as a clinical reagent ortherapeutic.

In certain aspects of the soluble fusion protein complexes of theinvention, the IL-15 polypeptide is an IL-15 variant having a differentamino acid sequence than native IL-15 polypeptide. The human IL-15polypeptide is referred to herein as huIL-15, hIL-15, huIL15, hIL15,IL-15 wild type (wt), and variants thereof are referred to using thenative amino acid, its position in the mature sequence and the variantamino acid. For example, huIL15N72D refers to human IL-15 comprising asubstitution of N to D at position 72. In one aspect, the IL-15 variantfunctions as an IL-15 agonist as demonstrated, e.g., by increasedbinding activity for the IL-15RβγC receptors compared to the nativeIL-15 polypeptide. Alternatively, the IL-15 variant functions as anIL-15 antagonist as demonstrated by e.g., decreased binding activity forthe IL-15RβγC receptors compared to the native IL-15 polypeptide.

Methods of enhancing immune function are carried out by a) contacting aplurality of cells with a soluble fusion protein complex of theinvention, wherein the plurality of cells further include immune cellsbearing the IL-15R chains recognized by the IL-15 domain, the IL-7Rchains recognized by the IL-7 domain or the IL-21R chains recognized bythe IL-21 domain, and b) inducing proliferation and activation theimmune cells via signaling of the IL-15R, IL-7R or IL-21R. In oneaspect, the method of enhancing immune function further includesactivation of the immune cells via signaling of a combination of atleast two or all of the IL-15R, IL-7R and IL-21R by the soluble fusionprotein complex. Exemplary methods for enhancing immune function includeinducing proliferation and activation of NK and T cells via signaling ofthe IL-15R, IL-12R and IL-18R by the soluble fusion protein complex.Such methods include proliferation and activation of NK and T cellsresulting in increased interferon gamma (IFN-γ) production.

Methods for killing a target cell are carried out by a) contacting aplurality of cells with a soluble fusion protein complex of theinvention, wherein the plurality of cells further include immune cellsbearing the IL-15R chains recognized by the IL-15 domain, the IL-7Rchains recognized by the IL-7 domain or the IL-21R chains recognized bythe IL-21 domain, and the target disease cells, b) inducingproliferation and activation of the immune cells via signaling of theIL-15R, IL-7R or IL-21R; and c) killing the target disease cells by theactivated immune cells. In one aspect, the method includes inducingproliferation and activation the immune cells via signaling of acombination of at least two or all of the IL-15R, IL-7R and IL-21R bythe soluble fusion protein complex. Exemplary methods include inducingproliferation and activation of NK and T cells via signaling of theIL-15R, IL-7R and IL-21R by the soluble fusion protein complex. Suchmethods include proliferation and activation of NK and T cells resultingin increased IFN-γ production.

The invention also provides methods for preventing or treating diseasein a patient, the method including the steps of: a) mixing immune cellsbearing IL-15R chains or checkpoint or signaling molecules with asoluble fusion protein complex of the invention, b) inducingproliferation and activation of the immune cells, c) administering (oradoptively transfer) to the patient the activated immune cells; and d)damaging or killing the disease cells via the activated immune cellssufficient to prevent or treat the disease in the patient. In oneaspect, the method includes proliferation and activation the immunecells via signaling of a combination of at least two or all of theIL-15R, IL-7R and IL-21R by the soluble fusion protein complex.Exemplary methods include inducing proliferation and activation of NKand T cells via signaling of the IL-15R, IL-7R and IL-21R by the solublefusion protein complex. Some aspects of the method include use of NK andT cells expressing chimeric antigen receptors (CAR NK and CAR T cells).In some embodiments of the invention, the patient is pretreated orpreconditioned to facilitate engraftment or survival of the adoptivelytransferred cells. Examples of preconditioning include treatment withcyclophosphamide and fludarabine. Additionally, the patient may betreated with agents that promote activation, survival or persistence ofthe adoptively transferred cells pre- and/or post-cell transfer.Examples of such treatment include use of IL-2, IL-15, ALT-803 (alsointerchangeably referred to herein as “N-803”) or otherimmunostimulatory agents. Other therapeutic approaches of known in thefield of adoptive cell therapy (i.e., including but not limited toallogeneic, autologous, haploidentical, DLI, stem cell, NK92-based andCAR NK therapies) may also be used in the methods herein.

Also provided are methods for preventing or treating disease in apatient, the method including the steps of: a) administering to thepatient a soluble fusion protein complex of the invention; b) inducingproliferation and activation of the immune cells in the patient; and c)damaging or killing the disease cells via the activated immune cellssufficient to prevent or treat the disease in the patient.

Administration of the fusion protein complexes of the invention inducesan immune response in a subject. For example, administration of thefusion protein complexes of the invention induces an immune responseagainst cells associated with neoplasia, infectious disease, senescentcell- or age-related diseases or autoimmune disease. In one aspect, thefusion protein complex of the invention increases immune cellproliferation, activation markers, cytotoxicity against target cells,and/or production of pro-inflammatory cytokines.

The invention provides methods of stimulating immune responses in amammal by administering to the mammal an effective amount of the solublefusion protein complex of the invention. The invention also providesmethods of suppressing immune responses in a mammal by administering tothe mammal an effective amount of the soluble fusion protein complex ofany one of the invention.

Methods for treating a neoplasia, infectious disease, senescent cell- orage-related diseases or autoimmune disease in a subject in need thereofare carried out by administering to a subject an effective amount ofexpanded and activated immune cells or a pharmaceutical compositioncomprising a soluble fusion protein complex described herein. Forexample, methods for treating solid or hematological malignancies in asubject in need thereof are carried out by administering to a subject aneffective amount of NK cells and T cells, and/or CAR NK and CAR T cellsexpanded ex vivo by the soluble fusion protein complex of the invention,thereby treating the malignancy. Exemplary soluble fusion proteincomplexes comprise the amino acid sequences set forth in SEQ ID NO: 2and SEQ ID NO: 4.

Suitable neoplasias for treatment with the methods described hereininclude a glioblastoma, prostate cancer, acute myeloid leukemia, B-cellneoplasm, multiple myeloma, B-cell lymphoma, B cell non-Hodgkin'slymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia, acutemyeloid leukemia, cutaneous T-cell lymphoma, T-cell lymphoma, a solidtumor, urothelial/bladder carcinoma, melanoma, lung cancer, renal cellcarcinoma, breast cancer, gastric and esophageal cancer, head and neckcancer, prostate cancer, pancreatic cancer, colorectal cancer, ovariancancer, non-small cell lung carcinoma, and squamous cell head and neckcarcinoma.

An exemplary infection for treatment using the methods described hereinincludes infections with human immunodeficiency virus (HIV) orcytomegalovirus (CMV). The methods described herein are also useful totreat bacterial infections (e.g., gram positive or gram negativebacteria) (See, e.g., Oleksiewicz et al. 2012. Arch Biochem Biophys.526:124-31, incorporated herein by reference).

Cell therapies of the invention comprise administration of an effectiveamount of expanded and activated immune cells. For example, an effectiveamount of expanded and activated NK or T cells is between 1×10⁴ cells/kgand 1×10¹⁰ cells/kg, e.g., 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, and1×10¹⁰ cells/kg, or such amounts that can be isolated by leukapheresis.Alternatively, expanded immune cells are administered as a fixed dose orbased on body surface area (i.e., per m²). Cells can be administeredafter ex vivo expansion or cryogenically preserved and administeredafter thawing (and washing as needed).

The pharmaceutical composition comprising a fusion protein complex isadministered in an effective amount. For example, an effective amount ofthe pharmaceutical composition is between about 1 μg/kg and 100 μg/kg,e.g., 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, or 100 μg/kg. Alternatively, TxM complex is administered asa fixed dose or based on body surface area (i.e., per m²).

The adoptively transferred immune cells or pharmaceutical compositioncomprising the fusion protein complex is administered at least one timeper month, e.g., twice per month, once per week, twice per week, onceper day, twice per day, every 8 hours, every 4 hours, every 2 hours, orevery hour. Suitable modes of administration for the adoptivelytransferred immune cells include systemic administration, intravenousadministration, or local administration. Suitable modes ofadministration for the pharmaceutical composition include systemicadministration, intravenous administration, local administration,subcutaneous administration, intramuscular administration, intratumoraladministration, inhalation, and intraperitoneal administration.

In an aspect, the present disclosure provides an isolated soluble fusionprotein complex comprising at least two soluble proteins, where thefirst soluble protein comprises an interleukin-15 (IL-15) polypeptidedomain and the second soluble protein comprises a soluble IL-15 receptoralpha sushi-binding domain (IL-15RαSu) fused to an immunoglobulin Fcdomain, where one of the first or second soluble protein furthercomprises an IL-7 binding domain or functional fragment thereof, whereone of the first or second soluble protein further comprises an IL-21binding domain or functional fragment thereof and wherein the IL-15domain of the first soluble protein binds to the IL-15RαSu domain of thesecond soluble protein to form a soluble fusion protein complex.

In an embodiment, the IL-15 polypeptide is an IL-15 variant comprisingan N72D mutation (IL-15N72D).

In an embodiment, the first soluble protein comprises the amino acidsequence set forth in SEQ ID NO: 2.

In an embodiment, the second soluble protein comprises the amino acidsequence set forth in SEQ ID NO: 4.

In an embodiment, a first soluble fusion protein complex may becovalently linked to a second soluble fusion protein complex.

In an embodiment, the first soluble fusion protein complex is covalentlylinked to the second soluble fusion protein complex by a disulfide bondlinking the Fc domain of the first soluble fusion protein complex to theFc domain of the second soluble fusion protein complex.

In an embodiment, the first or second soluble protein further comprisesa binding domain that recognizes a disease antigen.

In an embodiment, the first or second soluble protein further comprisesa binding domain that recognizes an immune checkpoint or signalingmolecule.

In an embodiment, the disease antigen is associated with a neoplasia,infectious disease or senescent cell- or age-related disease.

In an embodiment, the first soluble protein is encoded by the nucleicacid sequence set forth in SEQ ID NO: 1.

In an embodiment, the nucleic acid sequence further comprises apromoter, translation initiation signal, and leader sequence operablylinked to the sequence encoding the soluble protein.

In an embodiment, the second soluble protein may be encoded by thenucleic acid sequence set forth in SEQ ID NO: 3.

In an embodiment, the nucleic acid sequence further comprises apromoter, translation initiation signal, and leader sequence operablylinked to the sequence encoding the soluble protein.

In an embodiment, a DNA vector may comprise any of the above enumeratednucleic acid sequences.

In an embodiment, a method for enhancing immune function, the methodcomprising: a) contacting a plurality of cells with any of the abovesoluble fusion protein complexes, where the plurality of cells furthercomprises immune cells bearing the IL-15R chains recognized by the IL-15domain, the IL-7R chains recognized by the IL-7 domain and/or the IL-21Rchains recognized by the IL-21 domain, and b) inducing proliferation andactivation of the immune cells via signaling of the IL-15R, IL-7R and/orIL-21R.

In an aspect, the present disclosure provides a method for killing atarget cell, comprising: a) contacting a plurality of cells with any ofthe above soluble fusion protein complexes, where the plurality of cellsfurther include immune cells bearing the IL-15R chains recognized by theIL-15 domain, the IL-7R chains recognized by the IL-7 domain and/or theIL-21R chains recognized by the IL-21 domain, and the target diseasecells, b) inducing proliferation and activation of the immune cells viasignaling of the IL-15R, IL-7R and/or IL-21R, and c) killing the targetdisease cells by the expanded and activated immune cells.

In an embodiment, the target cells are tumor cells or infected cells.

In an aspect, the present disclosure provides a method of enhancingimmune responses in a subject, comprising: a) contacting a plurality ofcells with any of the above soluble fusion protein complexes, where theplurality of cells further include immune cells bearing the IL-15Rchains recognized by the IL-15 domain, the IL-7R chains recognized bythe IL-7 domain and/or the IL-21R chains recognized by the IL-21 domain,b) inducing proliferation and activation of the immune cells viasignaling of the IL-15R, IL-7R and/or IL-21R, c) administering (oradoptively transfer) to the patient the expanded and activated immunecells; and d) enhancing immune responses in the patient.

In an aspect, the present disclosure provides a method of preventing ortreating disease in a patient, comprising: a) contacting a plurality ofcells with a soluble fusion protein complex embodied herein, wherein theplurality of cells further includes immune cells bearing the IL-15Rchains recognized by the IL-15 domain, the IL-7R chains recognized bythe IL-7 domain and/or the IL-21R chains recognized by the IL-21 domain,b) inducing proliferation and activation of the immune cells viasignaling of the IL-15R, IL-7R and/or IL-21R, c) administering (oradoptively transfer) an effective amount of the expanded and activatedimmune cells to the patient, and d) damaging or killing the diseasecells via the expanded immune cells sufficient to prevent or treat thedisease in the patient.

In certain embodiments, a method of stimulating an immune response in asubject comprises isolating immune cells; contacting the immune cellswith a soluble fusion protein complex embodied herein; reinfusing theimmune cells into the subject; thereby, stimulating the immune responsein a subject. In certain embodiments, the immune cells compriseautologous, haplo-identical, haplotype matched or combinations thereof.In certain embodiments, the immune cells are derived from autologous orallogeneic stem cells. In certain embodiments, the immune cells compriseNK cells, T cells, stem cell memory T cells, activated NK (aNK) cells,chimeric antigen receptor-NK (CAR-NK) cells, chimeric antigen receptor-T(CAR-T) cells, or combinations thereof. In certain embodiments, one ormore adjuvants are optionally administered with the soluble fusionprotein complexes embodied herein.

In an embodiment, the disease is a neoplasia, infectious disease orsenescent cell- or age-related disease.

In an aspect, the present disclosure provides a method of enhancingimmune responses in a subject comprising administering to the subject aneffective amount of any of the above soluble fusion protein complexes.

In an aspect, the present disclosure provides a method for treating aneoplasia, infectious disease or senescent cell- or age-related diseasein a subject in need thereof comprising administering to said subject aneffective amount of a pharmaceutical composition comprising any of theabove soluble fusion protein complexes, thereby treating said neoplasia,infectious disease or senescent cell- or age-related disease.

In other aspects, a method of treating a subject having a neoplasia,infectious disease or senescent cell- or age-related disease, comprisesa) contacting immune cells with any of the above soluble fusion proteincomplexes, to induce proliferation and activation of the immune cell; b)administering (or adoptively transfer) an effective amount of theactivated immune cells to the subject, and c) damaging or killing thedisease cells via the activated immune cells sufficient to prevent ortreat the disease in the subject.

In an embodiment, the neoplasia is selected from the group consisting ofa glioblastoma, prostate cancer, hematological cancer, B-cell neoplasms,multiple myeloma, B-cell lymphoma, B cell non-Hodgkin lymphoma,Hodgkin's lymphoma, chronic lymphocytic leukemia, acute myeloidleukemia, cutaneous T-cell lymphoma, T-cell lymphoma, a solid tumor,urothelial/bladder carcinoma, melanoma, lung cancer, renal cellcarcinoma, breast cancer, gastric and esophageal cancer, prostatecancer, pancreatic cancer, colorectal cancer, ovarian cancer, non-smallcell lung carcinoma, and squamous cell head and neck carcinoma.

In another embodiment, the senescent cell- or age-related disease isselected from the group consisting of metabolic (obesity, diabetes),neurological (Alzheimer's and Parkinson's diseases), muscle, bone, andcartilage related (sarcopenia, osteoarthritis, kyphosis, herniateddiscs) or tissue dysfunction related (lung emphysema, cardiovascular andrenal diseases, and atherosclerosis) diseases.

In an embodiment, the immune cells are NK cells or cytokine inducedmemory like (CIML) NK cells.

In another embodiment, the immune cells are T cells or memory stem Tcells (T_(SCM)).

In an embodiment, the effective amounts of the expanded and activatedimmune cells are between 1×10⁴ cells/kg and 1×10¹⁰ cells/kg.

In an embodiment, the immune cells are administered at least one timeper week.

In an embodiment, the effective amount is between about 1 and 100 μg/kgsaid fusion protein complex.

In an embodiment, the fusion protein complex is administered at leastone time per week.

In an embodiment, the fusion protein complex increases immune cellproliferation, activation markers, cytotoxicity against target cells,and/or production of pro-inflammatory cytokines, including IFN-γ.

Preferably, the fusion protein complex increases serum levels ofinterferon gamma (IFN-γ), and/or stimulates CD4⁺ and CD8⁺ T cells and NKcells to kill diseased cells or tumor cells in a subject.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The terminology used herein is for thepurpose of describing particular embodiments only and is not intended tobe limiting of the invention.

The following references provide one of skill with a general definitionof many of the terms used in this invention: Singleton et al.,Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); TheCambridge Dictionary of Science and Technology (Walker ed., 1988); TheGlossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag(1991); and Hale & Marham, The Harper Collins Dictionary of Biology(1991). As used herein, the following terms have the meanings ascribedto them below, unless specified otherwise.

As used herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. Furthermore, to the extent that the terms “including”,“includes”, “having”, “has”, “with”, or variants thereof are used ineither the detailed description and/or the claims, such terms areintended to be inclusive in a manner similar to the term “comprising.”

Unless specifically stated or obvious from context, as used herein, theterms “a”, “an”, and “the” are understood to be singular or plural. Asused in this specification and the appended claims, the term “or” isgenerally employed in its sense including “and/or” unless the contentclearly dictates otherwise.

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. About can beunderstood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear fromcontext, all numerical values provided herein are modified by the termabout.

By “agent” is meant a peptide, nucleic acid molecule, or small compound.

By “ALT-803” or “N-803” is meant a complex comprising IL-15N72Dnoncovalently associated with a dimeric IL-15RαSu/Fc fusion protein andhaving immune stimulating activity. This complex is also referred to asIL-15 SA. In one embodiment, the IL-15N72D and/or IL-15RαSu/Fc fusionprotein comprises one, two, three, four or more amino acid variationsrelative to a reference sequence.

By “TxM” is meant a complex comprising an IL-15N72D:IL-15RαSu/Fcscaffold linked to a binding domain (FIGS. 1A, 1B). An exemplary TxM isan IL-15N72D:IL-15RαSu complex comprising fusions to IL-7 and IL-21cytokines.

By “ameliorate” is meant decrease, suppress, attenuate, diminish,arrest, or stabilize the development or progression of a disease.

By “analog” is meant a molecule that is not identical, but has analogousfunctional or structural features. For example, a polypeptide analogretains the biological activity of a corresponding naturally-occurringpolypeptide, while having certain biochemical modifications that enhancethe analog's function relative to a naturally occurring polypeptide.Such biochemical modifications could increase the analog's proteaseresistance, membrane permeability, or half-life, without altering, forexample, ligand binding. An analog may include an unnatural amino acid.“Analogs” in reference to nucleosides includes synthetic nucleosideshaving modified base moieties and/or modified sugar moieties, e.g.,described generally by Scheit, Nucleotide Analogs, John Wiley, New York,1980; Freier & Altmann, Nucl. Acid. Res., 1997, 25(22), 4429-4443,Toulmé, J. J., Nature Biotechnology 19:17-18 (2001); Manoharan M.,Biochemica et Biophysica Acta 1489:117-139(1999); Freier S. M., NucleicAcid Research, 25:4429-4443 (1997), Uhlman, E., Drug Discovery &Development, 3: 203-213 (2000), Herdewin P., Antisense & Nucleic AcidDrug Dev., 10:297-310 (2000)); 2′-O, 3′-C-linked[3.2.0]bicycloarabinonucleosides (see e.g. N. K Christiensen., et al.,J. Am. Chem. Soc., 120: 5458-5463 (1998). Such analogs include syntheticnucleosides designed to enhance binding properties, e.g., duplex ortriplex stability, specificity, or the like.

The invention includes antibodies or fragments of such antibodies, solong as they exhibit the desired biological activity. Also included inthe invention are chimeric antibodies, such as humanized antibodies.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source that is non-human. Humanization can beperformed, for example, using methods described in the art, bysubstituting at least a portion of a rodent complementarity-determiningregion for the corresponding regions of a human antibody.

The term “antibody” or “immunoglobulin” is intended to encompass bothpolyclonal and monoclonal antibodies. The preferred antibody is amonoclonal antibody reactive with the antigen. The term “antibody” isalso intended to encompass mixtures of more than one antibody reactivewith the antigen (e.g., a cocktail of different types of monoclonalantibodies reactive with the antigen). The term “antibody” is furtherintended to encompass whole antibodies, biologically functionalfragments thereof, single-chain antibodies, and genetically alteredantibodies such as chimeric antibodies comprising portions from morethan one species, bifunctional antibodies, antibody conjugates,humanized and human antibodies. Biologically functional antibodyfragments, which can also be used, are those peptide fragments derivedfrom an antibody that are sufficient for binding to the antigen.“Antibody” as used herein is meant to include the entire antibody aswell as any antibody fragments (e.g. F(ab′)₂, Fab′, Fab, Fv) capable ofbinding the epitope, antigen, or antigenic fragment of interest.

As used herein, the terms “associated with,” “conjugated,” “linked,”“attached,” and “tethered,” when used with respect to two or moremoieties, means that the moieties are physically associated or connectedwith one another, either directly (e.g. covalent bonding) or via one ormore additional moieties that serves as a linking agent, to form astructure that is sufficiently stable so that the moieties remainphysically associated under the conditions in which the structure isused, e.g., physiological conditions. In some embodiments, a sufficientnumber of weaker interactions can provide sufficient stability formoieties to remain physically associated under a variety of differentconditions.

By “binding to” a molecule is meant having a physicochemical affinityfor that molecule.

The term “binding domain” is intended to encompass an antibody, singlechain antibody, Fab, Fv, T-cell receptor binding domain, ligand bindingdomain, receptor binding domain, or other antigen-specific polypeptidesknown in the art.

As used herein, the term “biologically active moiety” or “effectormolecule” is meant a nucleic acid sequence, an amino acid sequence suchas a protein, polypeptide or peptide; a sugar or polysaccharide; a lipidor a glycolipid, glycoprotein, or lipoprotein that can produce thedesired effects as discussed herein. Effector molecules also includechemical agents. Also contemplated are effector molecule nucleic acidsencoding a biologically active or effector protein, polypeptide, orpeptide. Thus, suitable molecules include regulatory factors, enzymes,antibodies, or drugs as well as DNA, RNA, and oligonucleotides. Thebiologically active polypeptides or effector molecule can benaturally-occurring or it can be synthesized from known components,e.g., by recombinant or chemical synthesis and can include heterologouscomponents. A biologically active polypeptide or effector molecule isgenerally between about 0.1 to 100 KD or greater up to about 1000 KD,preferably between about 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 30 and 50 KD asjudged by standard molecule sizing techniques such as centrifugation orSDS-polyacrylamide gel electrophoresis. Desired effects of the inventioninclude, but are not limited to, for example, forming a fusion proteincomplex of the invention with increased binding activity, killing atarget cell, e.g. either to induce cell proliferation or cell death,initiate an immune response, in preventing or treating a disease, or toact as a detection molecule for diagnostic purposes. For such detection,an assay could be used, for example an assay that includes sequentialsteps of culturing cells to proliferate same, and contacting the cellswith a fusion complex of the invention and then evaluating whether thefusion complex inhibits further development of the cells.

Covalently linking the effector molecule to the fusion protein complexesof the invention in accordance with the invention provides a number ofsignificant advantages. Fusion protein complexes of the invention can beproduced that contain a single effector molecule, including a peptide ofknown structure. Additionally, a wide variety of effector molecules canbe produced in similar DNA vectors. That is, a library of differenteffector molecules can be linked to the fusion protein complexes forrecognition of infected or diseased cells. Further, for therapeuticapplications, rather than administration of the fusion protein complexof the invention to a subject, a DNA expression vector coding for thefusion protein complex can be administered for in vivo expression of thefusion protein complex. Such an approach avoids costly purificationsteps typically associated with preparation of recombinant proteins andavoids the complexities of antigen uptake and processing associated withconventional approaches.

As noted, components of the fusion proteins disclosed herein, e.g.,effector molecule such as cytokines, chemokines, growth factors, proteintoxins, immunoglobulin domains or other bioactive molecules and anypeptide linkers, can be organized in nearly any fashion provided thatthe fusion protein has the function for which it was intended. Inparticular, each component of the fusion protein can be spaced fromanother component by at least one suitable peptide linker sequence ifdesired. Additionally, the fusion proteins may include tags, e.g., tofacilitate modification, identification and/or purification of thefusion protein. More specific fusion proteins are in the Examplesdescribed below.

The term “chimeric antigen receptor” or “CAR” as used herein refers toan antigen-binding domain that is fused to an intracellular signalingdomain capable of activating or stimulating an immune cell, and incertain embodiments, the CAR also comprises a transmembrane domain. Incertain embodiments the CAR's extracellular antigen-binding domain iscomposed of a single chain variable fragment (scFv) derived from fusingthe variable heavy and light regions of a murine or humanized monoclonalantibody. Alternatively, scFvs may be used that are derived from Fab's(instead of from an antibody, e.g., obtained from Fab libraries). Invarious embodiments, the scFv is fused to the transmembrane domain andthen to the intracellular signaling domain. “First-generation” CARsinclude those that solely provide CD3 signals upon antigen binding,“Second-generation” CARs include those that provide both co-stimulation(e.g., CD28 or CD137) and activation (CD3). “Third-generation” CARsinclude those that provide multiple co-stimulation (e.g. CD28 and CD137)and activation (CD3). A fourth generation of CARs have been described,CAR T cells redirected for cytokine killing (TRUCKS) where the vectorcontaining the CAR construct possesses a cytokine cassette. When the CARis ligated, the CAR T cell deposits a pro-inflammatory cytokine into thetumor lesion. A CAR-T cell is a T cell that expresses a chimeric antigenreceptor. A CAR-NK cell is an NK cell expressing a chimeric antigenreceptor. The chimeric antigen receptors (CARs), have anantigen-specific extracellular domain coupled to an intracellular domainthat directs the cell to perform a specialized function upon binding ofan antigen to the extracellular domain. The terms “artificial T-cellreceptor,” “chimeric T-cell receptor,” and “chimeric immunoreceptor” mayeach be used interchangeably herein with the term “chimeric antigenreceptor.” Chimeric antigen receptors are distinguished from otherantigen binding agents by their ability to both bind MHC-independentantigen and transduce activation signals via their intracellular domain.

“Detect” refers to identifying the presence, absence or amount of theanalyte to be detected.

By “disease” is meant any condition or disorder that damages orinterferes with the normal function of a cell, tissue, or organ.Examples of diseases include neoplasia, autoimmune diseases, viralinfections, and senescent cell- and age-related disease.

By the terms “effective amount” and “therapeutically effective amount”of a formulation or formulation component is meant a sufficient amountof the formulation or component, alone or in a combination, to providethe desired effect. For example, by “an effective amount” is meant anamount of a compound, alone or in a combination, required to amelioratethe symptoms of a disease relative to an untreated patient. Theeffective amount of active compound(s) used to practice the presentinvention for therapeutic treatment of a disease varies depending uponthe manner of administration, the age, body weight, and general healthof the subject. Ultimately, the attending physician or veterinarian willdecide the appropriate amount and dosage regimen. Such amount isreferred to as an “effective” amount.

By “fragment” is meant a portion of a polypeptide or nucleic acidmolecule. This portion contains, preferably, at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the referencenucleic acid molecule or polypeptide. For example, a fragment maycontain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500,600, 700, 800, 900, or 1000 nucleotides or amino acids. However, theinvention also comprises polypeptides and nucleic acid fragments, solong as they exhibit the desired biological activity of the full lengthpolypeptides and nucleic acid, respectively. A nucleic acid fragment ofalmost any length is employed. For example, illustrative polynucleotidesegments with total lengths of about 10,000, about 5,000, about 3,000,about 2,000, about 1,000, about 500, about 200, about 100, about 50 basepairs in length (including all intermediate lengths) are included inmany implementations of this invention. Similarly, a polypeptidefragment of almost any length is employed. For example, illustrativepolypeptide segments with total lengths of about 10,000, about 5,000,about 3,000, about 2,000, about 1,000, about 5,000, about 1,000, about500, about 200, about 100, or about 50 amino acids in length (includingall intermediate lengths) are included in many implementations of thisinvention.

As used herein, the term “immune cells” generally includes white bloodcells (leukocytes) which are derived from hematopoietic stem cells (HSC)produced in the bone marrow “Immune cells” includes, e.g., lymphocytes(T cells, B cells, natural killer (NK) cells) and myeloid-derived cells(neutrophil, eosinophil, basophil, monocyte, macrophage, dendriticcells).

The term “immune effector cell,” as used herein, refers to a cell thatis involved in an immune response, e.g., in the promotion of an immuneeffector response. Examples of immune effector cells include T cells,e.g., alpha/beta T cells and gamma/delta T cells, B cells, naturalkiller (NK) cells, natural killer T (NK-T) cells, mast cells, andmyeloid-derived phagocytes. “Immune effector function or immune effectorresponse,” as that term is used herein, refers to function or response,e.g., of an immune effector cell, that enhances or promotes an immuneattack of a target cell. For example, an immune effector function orresponse refers a property of a T or NK cell that promotes killing orthe inhibition of growth or proliferation, of a target cell. In the caseof a T cell, primary stimulation and co-stimulation are examples ofimmune effector function or response.

The terms “isolated”, “purified”, or “biologically pure” refer tomaterial that is free to varying degrees from components which normallyaccompany it as found in its native state. “Isolate” denotes a degree ofseparation from original source or surroundings. “Purify” denotes adegree of separation that is higher than isolation.

A “purified” or “biologically pure” protein is sufficiently free ofother materials such that any impurities do not materially affect thebiological properties of the protein or cause other adverseconsequences. That is, a nucleic acid or peptide of this invention ispurified if it is substantially free of cellular material, viralmaterial, or culture medium when produced by recombinant DNA techniques,or chemical precursors or other chemicals when chemically synthesized.Purity and homogeneity are typically determined using analyticalchemistry techniques, for example, polyacrylamide gel electrophoresis orhigh performance liquid chromatography. The term “purified” can denotethat a nucleic acid or protein gives rise to essentially one band in anelectrophoretic gel. For a protein that can be subjected tomodifications, for example, phosphorylation or glycosylation, differentmodifications may give rise to different isolated proteins, which can beseparately purified.

Similarly, by “substantially pure” is meant a nucleotide or polypeptidethat has been separated from the components that naturally accompany it.Typically, the nucleotides and polypeptides are substantially pure whenthey are at least 60%, 70%, 80%, 90%, 95%, or even 99%, by weight, freefrom the proteins and naturally-occurring organic molecules with theyare naturally associated.

By “isolated nucleic acid” is meant a nucleic acid that is free of thegenes which flank it in the naturally-occurring genome of the organismfrom which the nucleic acid is derived. The term covers, for example:(a) a DNA which is part of a naturally occurring genomic DNA molecule,but is not flanked by both of the nucleic acid sequences that flank thatpart of the molecule in the genome of the organism in which it naturallyoccurs; (b) a nucleic acid incorporated into a vector or into thegenomic DNA of a prokaryote or eukaryote in a manner, such that theresulting molecule is not identical to any naturally occurring vector orgenomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment,a fragment produced by polymerase chain reaction (PCR), or a restrictionfragment; and (d) a recombinant nucleotide sequence that is part of ahybrid gene, i.e., a gene encoding a fusion protein. Isolated nucleicacid molecules according to the present invention further includemolecules produced synthetically, as well as any nucleic acids that havebeen altered chemically and/or that have modified backbones. Forexample, the isolated nucleic acid is a purified cDNA or RNApolynucleotide. Isolated nucleic acid molecules also include messengerribonucleic acid (mRNA) molecules.

By an “isolated polypeptide” is meant a polypeptide of the inventionthat has been separated from components that naturally accompany it.Typically, the polypeptide is isolated when it is at least 60%, byweight, free from the proteins and naturally-occurring organic moleculeswith which it is naturally associated. Preferably, the preparation is atleast 75%, more preferably at least 90%, and most preferably at least99%, by weight, a polypeptide of the invention. An isolated polypeptideof the invention may be obtained, for example, by extraction from anatural source, by expression of a recombinant nucleic acid encodingsuch a polypeptide; or by chemically synthesizing the protein. Puritycan be measured by any appropriate method, for example, columnchromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

By “marker” is meant any protein or polynucleotide having an alterationin expression level or activity that is associated with a disease ordisorder.

By “neoplasia” is meant a disease or disorder characterized by excessproliferation or reduced apoptosis. Illustrative neoplasms for which theinvention can be used include, but are not limited to leukemias (e.g.,acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia,acute myeloblastic leukemia, acute promyelocytic leukemia, acutemyelomonocytic leukemia, acute monocytic leukemia, acuteerythroleukemia, chronic leukemia, chronic myelocytic leukemia, chroniclymphocytic leukemia), polycythemia vera, lymphoma (Hodgkin's disease,non-Hodgkin's disease), Waldenstrom's macroglobulinemia, heavy chaindisease, and solid tumors such as sarcomas and carcinomas (e.g.,fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, nile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterinecancer, testicular cancer, lung carcinoma, small cell lung carcinoma,bladder carcinoma, epithelial carcinoma, glioma, glioblastomamultiforme, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma,pinealoma, hemangioblastoma, acoustic neuroma, oligodenroglioma,schwannoma, meningioma, melanoma, neuroblastoma, and retinoblastoma). Inparticular embodiments, the neoplasia is multiple myeloma, beta-celllymphoma, urothelial/bladder carcinoma or melanoma. As used herein,“obtaining” as in “obtaining an agent” includes synthesizing,purchasing, or otherwise acquiring the agent.

As used herein, the terms “nucleic acid sequence”, “polynucleotide,” and“gene” are used interchangeably throughout the specification and includecomplementary DNA (cDNA), linear or circular oligomers or polymers ofnatural and/or modified monomers or linkages, includingdeoxyribonucleosides, ribonucleosides, substituted and alpha-anomericforms thereof, peptide nucleic acids (PNA), locked nucleic acids (LNA),phosphorothioate, methylphosphonate, and the like. Polynucleotidesinclude, but are not limited to, all nucleic acid sequences which areobtained by any means available in the art, including, withoutlimitation, recombinant means, i.e., the cloning of nucleic acidsequences from a recombinant library or a cell genome, using ordinarycloning technology and PCR™, and the like, and by synthetic means.

The nucleic acid sequences may be “chimeric,” that is, composed ofdifferent regions. In the context of this invention “chimeric” compoundsare oligonucleotides, which contain two or more chemical regions, forexample, DNA region(s), RNA region(s), PNA region(s) etc. Each chemicalregion is made up of at least one monomer unit, i.e., a nucleotide.These sequences typically comprise at least one region wherein thesequence is modified in order to exhibit one or more desired properties.

Nucleic acid molecules useful in the methods of the invention includeany nucleic acid molecule that encodes a polypeptide of the invention ora fragment thereof. Such nucleic acid molecules need not be 100%identical with an endogenous nucleic acid sequence, but will typicallyexhibit substantial identity. Polynucleotides having “substantialidentity” to an endogenous sequence are typically capable of hybridizingwith at least one strand of a double-stranded nucleic acid molecule.Nucleic acid molecules useful in the methods of the invention includeany nucleic acid molecule that encodes a polypeptide of the invention ora fragment thereof. Such nucleic acid molecules need not be 100%identical with an endogenous nucleic acid sequence, but will typicallyexhibit substantial identity. Polynucleotides having “substantialidentity” to an endogenous sequence are typically capable of hybridizingwith at least one strand of a double-stranded nucleic acid molecule. By“hybridize” is meant pair to form a double-stranded molecule betweencomplementary polynucleotide sequences (e.g., a gene described herein),or portions thereof, under various conditions of stringency. (See, e.g.,Wahl, G. M., and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel,A. R. (1987) Methods Enzymol. 152:507).

For example, stringent salt concentration will ordinarily be less thanabout 750 mM NaCl and 75 mM trisodium citrate, preferably less thanabout 500 mM NaCl and 50 mM trisodium citrate, and more preferably lessthan about 250 mM NaCl and 25 mM trisodium citrate. Low stringencyhybridization can be obtained in the absence of organic solvent, e.g.,formamide, while high stringency hybridization can be obtained in thepresence of at least about 35% formamide, and more preferably at leastabout 50% formamide. Stringent temperature conditions will ordinarilyinclude temperatures of at least about 30° C., more preferably of atleast about 37° C., and most preferably of at least about 42° C. Varyingadditional parameters, such as hybridization time, the concentration ofdetergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion orexclusion of carrier DNA, are well known to those skilled in the art.Various levels of stringency are accomplished by combining these variousconditions as needed. In a preferred: embodiment, hybridization willoccur at 30° C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. Ina more preferred embodiment, hybridization will occur at 37° C. in 500mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100.mu.g/ml denatured salmon sperm DNA (ssDNA). In a most preferredembodiment, hybridization will occur at 42° C. in 250 mM NaCl, 25 mMtrisodium citrate, 1% SDS, 50% formamide, and 200 μg/ml ssDNA. Usefulvariations on these conditions will be readily apparent to those skilledin the art.

For most applications, washing steps that follow hybridization will alsovary in stringency. Wash stringency conditions can be defined by saltconcentration and by temperature. As above, wash stringency can beincreased by decreasing salt concentration or by increasing temperature.For example, stringent salt concentration for the wash steps willpreferably be less than about 30 mM NaCl and 3 mM trisodium citrate, andmost preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.Stringent temperature conditions for the wash steps will ordinarilyinclude a temperature of at least about 25° C., more preferably of atleast about 42° C., and even more preferably of at least about 68° C. Ina preferred embodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, washsteps will occur at 42 C in 15 mM NaCl, 1.5 mM trisodium citrate, and0.1% SDS. In a more preferred embodiment, wash steps will occur at 68°C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additionalvariations on these conditions will be readily apparent to those skilledin the art. Hybridization techniques are well known to those skilled inthe art and are described, for example, in Benton and Davis (Science196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology,Wiley Interscience, New York, 2001); Berger and Kimmel (Guide toMolecular Cloning Techniques, 1987, Academic Press, New York); andSambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, New York.

As used herein, “nucleoside” includes the natural nucleosides, including2′-deoxy and 2′-hydroxyl forms, e.g., as described in Kornberg andBaker, DNA Replication, 2nd Ed. (Freeman, San Francisco, 1992).

By “senescent cell-related disease” or age-related disease” is meant adisease or disorder selected from the group consisting of metabolic(obesity, diabetes), neurological (Alzheimer's and Parkinson'sdiseases), muscle, bone, and cartilage related (sarcopenia,osteoarthritis, kyphosis, herniated discs) or tissue dysfunction related(lung emphysema, cardiovascular and renal diseases, and atherosclerosis)diseases.

By “reduces” is meant a negative alteration of at least 5%, 10%, 25%,50%, 75%, or 100%.

By “reference” is meant a standard or control condition.

A “reference sequence” is a defined sequence used as a basis forsequence comparison. A reference sequence may be a subset of or theentirety of a specified sequence; for example, a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence. For polypeptides, the length of the reference polypeptidesequence will generally be at least about 16 amino acids, preferably atleast about 20 amino acids, more preferably at least about 25 aminoacids, and even more preferably about 35 amino acids, about 50 aminoacids, or about 100 amino acids. For nucleic acids, the length of thereference nucleic acid sequence will generally be at least about 50nucleotides, preferably at least about 60 nucleotides, more preferablyat least about 75 nucleotides, and even more preferably about 100nucleotides or about 300 nucleotides or any integer thereabout ortherebetween.

By “specifically binds” is meant a compound or antibody that recognizesand binds a polypeptide of the invention, but which does notsubstantially recognize and bind other molecules in a sample, forexample, a biological sample, which naturally includes a polypeptide ofthe invention.

By “substantially identical” is meant a polypeptide exhibiting at least85% identity to a reference amino acid sequence (for example, any one ofthe amino acid sequences described herein). Preferably, such a sequenceis at least 90%, more preferably 95% or even 99% identical at the aminoacid level to the sequence used for comparison.

Sequence identity is typically measured using sequence analysis software(for example, Sequencher, Gene Codes Corporation, 775 Technology Drive,Ann Arbor, Mich.; Vector NTI, Life Technologies, 3175 Staley Rd. GrandIsland, N.Y.). Such software matches identical or similar sequences byassigning degrees of homology to various substitutions, deletions,and/or other modifications. Conservative substitutions typically includesubstitutions within the following groups: glycine, alanine; valine,isoleucine, leucine; aspartic acid, glutamic acid, asparagine,glutamine; serine, threonine; lysine, arginine; and phenylalanine,tyrosine. In an exemplary approach to determining the degree ofidentity, a BLAST program may be used, with a probability score betweene⁻³ and e⁻¹⁰⁰ indicating a closely related sequence.

By “subject” is meant a mammal, including, but not limited to, a humanor non-human mammal, such as a bovine, equine, canine, ovine, or feline.The subject is preferably a mammal in need of such treatment, e.g., asubject that has been diagnosed with B cell lymphoma or a predispositionthereto. The mammal is any mammal, e.g., a human, a primate, a mouse, arat, a dog, a cat, a horse, as well as livestock or animals grown forfood consumption, e.g., cattle, sheep, pigs, chickens, and goats. In apreferred embodiment, the mammal is a human.

Ranges provided herein are understood to be shorthand for all of thevalues within the range. For example, a range of 1 to 50 is understoodto include any number, combination of numbers, or sub-range from thegroup consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

The terms “treating” and “treatment” as used herein refer to theadministration of an agent or formulation to a clinically symptomaticindividual afflicted with an adverse condition, disorder, or disease, soas to effect a reduction in severity and/or frequency of symptoms,eliminate the symptoms and/or their underlying cause, and/or facilitateimprovement or remediation of damage. It will be appreciated that,although not precluded, treating a disorder or condition does notrequire that the disorder, condition or symptoms associated therewith becompletely eliminated. Agents or formulations used in treatment maycomprise cells or tissues.

Treatment of patients with neoplasia may include any of the following:Adjuvant therapy (also called adjunct therapy or adjunctive therapy) todestroy residual tumor cells that may be present after the known tumoris removed by the initial therapy (e.g. surgery), thereby preventingpossible cancer reoccurrence; neoadjuvant therapy given prior to thesurgical procedure to shrink the cancer; induction therapy to cause aremission, typically for acute leukemia; consolidation therapy (alsocalled intensification therapy) given once a remission is achieved tosustain the remission; maintenance therapy given in lower or lessfrequent doses to assist in prolonging a remission; first line therapy(also called standard therapy); second (or 3rd, 4th, etc.) line therapy(also called salvage therapy) is given if a disease has not responded orreoccurred after first line therapy; and palliative therapy (also calledsupportive therapy) to address symptom management without expecting tosignificantly reduce the cancer.

The terms “preventing” and “prevention” refer to the administration ofan agent or composition to a clinically asymptomatic individual who issusceptible or predisposed to a particular adverse condition, disorder,or disease, and thus relates to the prevention of the occurrence ofsymptoms and/or their underlying cause.

The term “variant,” when used in the context of a polynucleotidesequence, may encompass a polynucleotide sequence related to a wild typegene. This definition may also include, for example, “allelic,”“splice,” “species,” or “polymorphic” variants. A splice variant mayhave significant identity to a reference molecule, but will generallyhave a greater or lesser number of polynucleotides due to alternatesplicing of exons during mRNA processing. The corresponding polypeptidemay possess additional functional domains or an absence of domains.Species variants are polynucleotide sequences that vary from one speciesto another. Of particular utility in the invention are variants of wildtype target gene products. Variants may result from at least onemutation in the nucleic acid sequence and may result in altered mRNAs orin polypeptides whose structure or function may or may not be altered.Any given natural or recombinant gene may have none, one, or manyallelic forms. Common mutational changes that give rise to variants aregenerally ascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence. Theresulting polypeptides generally will have significant amino acididentity relative to each other. A polymorphic variant is a variation inthe polynucleotide sequence of a particular gene between individuals ofa given species. Polymorphic variants also may encompass “singlenucleotide polymorphisms” (SNPs) or single base mutations in which thepolynucleotide sequence varies by one base. The presence of SNPs may beindicative of, for example, a certain population with a propensity for adisease state, that is susceptibility versus resistance.

As used herein, “variant” of polypeptides refers to an amino acidsequence that is altered by one or more amino acid residues. The variantmay have “conservative” changes, wherein a substituted amino acid hassimilar structural or chemical properties (e.g., replacement of leucinewith isoleucine). More rarely, a variant may have “nonconservative”changes (e.g., replacement of glycine with tryptophan). Analogous minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted, or deleted without abolishing biological activity may be foundusing computer programs well known in the art, for example, LASERGENEsoftware (DNASTAR).

The recitation of a listing of chemical groups in any definition of avariable herein includes definitions of that variable as any singlegroup or combination of listed groups. The recitation of an embodimentfor a variable or aspect herein includes that embodiment as any singleembodiment or in combination with any other embodiments or portionsthereof.

Any compositions or methods provided herein can be combined with one ormore of any of the other compositions and methods provided herein.

The transitional term “comprising,” which is synonymous with“including,” “containing,” or “characterized by,” is inclusive oropen-ended and does not exclude additional, unrecited elements or methodsteps. By contrast, the transitional phrase “consisting of” excludes anyelement, step, or ingredient not specified in the claim. Thetransitional phrase “consisting essentially of” limits the scope of aclaim to the specified materials or steps “and those that do notmaterially affect the basic and novel characteristic(s)” of the claimedinvention.

Any genes, gene names, gene products or peptides disclosed herein areintended to correspond to homologs from any species for which thecompositions and methods disclosed herein are applicable. Thus, theterms include, but are not limited to genes and gene products fromhumans and mice. It is understood that when a gene or gene product froma particular species is disclosed, this disclosure is intended to beexemplary only, and is not to be interpreted as a limitation unless thecontext in which it appears clearly indicates. Thus, for example, forthe genes disclosed herein, which in some embodiments relate tomammalian nucleic acid and amino acid sequences are intended toencompass homologous and/or orthologous genes and gene products fromother animals including, but not limited to other mammals, fish,amphibians, reptiles, and birds. In preferred embodiments, the genes,nucleic acid sequences or peptides are human.

Genbank and NCBI submissions indicated by accession number cited hereinare incorporated herein by reference. All other published references,documents, manuscripts and scientific literature cited herein areincorporated herein by reference. In the case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims. Unless otherwise defined, all technical and scientific termsused herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Althoughmethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present invention,suitable methods and materials are described below. All publishedforeign patents and patent applications cited herein are incorporatedherein by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1A is a schematic diagram illustrating a TxM fusion protein complexcomprising the IL-15N72D:IL-15RαSu/Fc scaffold fused to IL-7 (FIG. 1B)and IL-21 (FIG. 1C) binding domains (IL7-IL15N72D:IL21-IL15RαSuFc). Insome cases, the dimeric IL-15RαSu/Fc fusion complexes comprise one ortwo IL-15N72D fusion protein proteins.

FIG. 2A is a line graph showing the chromatographic profile ofhIL7/IL21/TxM protein-containing cell culture supernatant followingbinding and elution on a Protein A resin. FIG. 2B is a line graphshowing the chromatographic profile of Protein A-purified hIL7/IL21/TxMfusion protein complex following elution on a preparative size exclusioncolumn. FIG. 2C is a line graph showing the chromatographic profile ofProtein A/SEC-purified hIL7/IL21/TxM fusion protein complex followingelution on an analytical size exclusion column, demonstrating separationof monomeric multiprotein hIL7/IL21/TxM fusion protein complexes fromprotein aggregates.

FIG. 3A shows photographs of 1) a Western blot to detect protein bandscontaining human IgG Fc domains (left panel) and 2) CoomassieBlue-stained sodium dodecyl sulfate polyacrylamide gel (4-12%)electrophoresis (SDS-PAGE) analysis (right panel) of the hIL7/IL21/TxMfusion protein complex following disulfide bond reduction. FIG. 3B showsphotographs of 1) a Western blot to detect protein bands containinghuman IL-15 domains (left panel) and 2) Coomassie Blue—sodium dodecylsulfate polyacrylamide gel (4-12%) electrophoresis (SDS-PAGE) analysis(right panel) of the hIL7/IL21/TxM fusion protein complex followingdisulfide bond reduction. For each type of analysis, lane 1 is the NovexSharp protein standard, lanes 2 & 3: purified hIL7/IL21/TxM Lot20170714, and lane 4: ALT-803 (IL-15N72D:IL-15RαSu/Fc complex) control.

FIG. 4 shows a photograph of Coomassie Blue-stained SDS-PAGE analysis ofALT-803 (control) (lanes 2 & 3) and hIL7/IL21/TxM fusion protein complex(lanes 4 & 5) following disulfide bond reduction. The protein samples inlanes 3 & 5 had been deglycosylated with the Protein Deglycosylation MixII (New England BioLabs) according to manufacturer's instructions. Lane1 is the Novex Sharp protein standard, lane 2: ALT-803, lane 3:deglycosylated ALT-803, lane 4: hIL7/IL21/TxM Lot 20170714, lane 5:deglycosylated hIL7/IL21/TxM Lot 20170714, and lane 6: deglycosylationreaction buffer (containing deglycosylase enzymes).

FIG. 5 is a line graph showing the binding activity of the hIL7/IL21/TxMfusion protein complex to antibodies specific to human IL-15 and humanIgG.

FIGS. 6A-6E are a series of line graphs depicting the binding activityof the hIL7/IL21/TxM fusion protein complex to antibodies specific tohuman IgG (GAH) (FIG. 6A), IL15 (FIG. 6B), IL7 and IL15 (FIG. 6C), IL21and IL15 (FIG. 6D) and IL7 and IL21 (FIG. 6E).

FIG. 7 is a line graph illustrating the proliferation of IL-15-dependent32D13 cells mediated by hIL7/IL21/TxM fusion protein complex compared toALT-803.

FIG. 8A is a bar graph showing IL-7 biological activity of hIL7/IL21/TxMfusion protein complex, a combination of recombinant IL-7, IL-21 andALT-803 (IL-7+IL-21+ALT-803), and recombinant IL-7 alone by measuringphosphorylation of Stat5 in mouse 2E8 cells by flow cytometry. Datarepresent the mean fluorescence intensity (MFI) fold change. FIG. 8B isa histogram showing IL-7 biological activity of hIL7/IL21/TxM fusionprotein complex, a combination of recombinant IL-17, IL-21 and ALT-803(IL-7+IL-21+ALT-803), and recombinant IL-7 alone compared to mediacontrol by measuring phosphorylation of Stat5 in mouse 2E8 cells by flowcytometry. FIG. 8C is a bar graph showing IL-21 biological activity ofhIL7/IL21/TxM fusion protein complex, a combination of recombinant IL-7,IL-21 and ALT-803 (IL-7+IL-21+ALT-803), and recombinant IL-21 alone bymeasuring phosphorylation of Stat3 in purified human T cells by flowcytometry. Data represent the MFI fold change. FIG. 8D is a histogramshowing IL-21 biological activity of hIL7/IL21/TxM fusion proteincomplex, a combination of recombinant IL-17, IL-21 and ALT-803(IL-7+IL-21+ALT-803), and recombinant IL-21 alone compared to mediacontrol by measuring phosphorylation of Stat3 in purified human T cellsby flow cytometry. FIG. 8E is a bar graph showing IL-15 biologicalactivity of hIL7/IL21/TxM fusion protein complex, a combination ofrecombinant IL-7, IL-21 and ALT-803 (IL-7+IL-21+ALT-803), and ALT-803alone by measuring phosphorylation of Stat5 in 32D3 cells by flowcytometry. Data represent the MFI fold changes. FIG. 8F is a histogramshowing IL-15 biological activity of hIL7/IL21/TxM fusion proteincomplex, a combination of recombinant IL-17, IL-21 and ALT-803(IL-7+IL-21+ALT-803), and ALT-803 alone compared to media control byphosphorylation of Stat5 in 32D3 cells by flow cytometry.

FIG. 9 is a bar graph showing IFN-γ production by purified human naïve Tcells following stimulation with hIL7/IL21/TxM fusion protein complexcompared to stimulation with combination of recombinant IL-7, IL-21 andALT-803 (IL-7+IL-21+ALT-803). IFN-γ production was measured by ELISA.

FIGS. 10A and 10B are bar graphs showing proliferation of purified humannaïve T cells from two donors (donor A, FIG. 10A; donor B, FIG. 10B)following stimulation for various times with hIL7/IL21/TxM fusionprotein complex or a combination of recombinant IL-7, IL-21 and ALT-803(IL-7+IL-21+ALT-803), compared to media control. Proliferation wasmeasured using Presto Blue Assay. FIG. 10C is a bar graph showingaverage human T cell proliferation at the 72 hr time-point for the datashown in FIGS. 10A and 10B.

FIG. 11 are dot-plots showing proliferation of purified CSFE-labeledhuman T cells following stimulation with hIL7/IL21/TxM fusion proteincomplex or a combination of recombinant IL-7, IL-21 and ALT-803(IL-7+IL-21+ALT-803), compared to media control. Proliferation wasassessed by dilution of CFSE signal following cell division.

FIG. 12 is a bar graph showing the expansion of purified human CD8⁺ Tcells following incubation in media containing hIL7/IL21/TxM fusionprotein complex; compared to IL-7+IL-21+ALT-803, ALT-803 alone orcontrol media. Incubation with hIL7/IL21/TxM resulted in betterexpansion of CD8⁺ T cells than was observed with IL-7+IL-21+ALT-803combination treatment.

FIG. 13 are density plots showing the proliferation and phenotype ofpurified human CD8⁺ T cells following incubation in media containinghIL7/IL21/TxM fusion protein complex; compared to IL-7+IL-21+ALT-803,ALT-803 alone or control media. Incubation with hIL7/IL21/TxM resultedin greater proliferation of CD8⁺ T cells, particular m than was observedwith IL-7+IL-21+ALT-803 combination treatment.

FIGS. 14A-14D depict bar graphs showing the expansion of purified humanCD8⁺ T cell subsets following incubation in media containinghIL7/IL21/TxM fusion protein complex; compared to control media (US),IL-7+IL-21, IL-7+ALT-803, IL-21+ALT-803, or IL-7+IL-21+ALT-803combinations. Incubation with hIL7/IL21/TxM resulted in expansion ofnaïve (FIG. 14A), central memory (FIG. 14B), effector memory (FIG. 14C)and memory stem CD8⁺ (FIG. 14D) T cell subsets compared to mediacontrols and better expansion of central memory and effector memory CD8⁺T cell subsets than any other combination of individual cytokines.

FIG. 15 shows line graphs (flow cytometry histograms) showing theproliferation of purified human CD8⁺ T cell subsets following incubationin media containing hIL7/IL21/TxM fusion protein complex, compared tocontrol media (US), IL-7+IL-21, IL-7+ALT-803, IL-21+ALT-803, orIL-7+IL-21+ALT-803 combinations. Incubation with hIL7/IL21/TxM resultedin proliferation of naïve, central memory, effector memory and memorystem CD8⁺ T cell subsets compared to media controls. Greaterproliferation of central memory and effector memory CD8⁺ T cell subsetswas seen with hIL7/IL21/TxM than any other combination of individualcytokines.

FIG. 16 is a line graph illustrating the proliferation of purified humanNK cells mediated by hIL7/IL21/TxM fusion protein complex compared toALT-803. The lines represent NK cells isolated from different donors.

FIG. 17 are density plots showing the phenotype of purified human NKcells following incubation in media containing hIL7/IL21/TxM fusionprotein complex; compared to IL-7+IL-21+ALT-803, ALT-803 alone orhIL18/IL12/TxM fusion protein complex containing media.

FIG. 18 are bar graphs showing levels of K562 target cell killingmediated by purified human NK cells following incubation in mediacontaining hIL7/IL21/TxM fusion protein complex; compared toIL-7+IL-21+ALT-803, ALT-803 alone or hIL18/IL12/TxM fusion proteincomplex containing media.

FIG. 19 are bar graphs showing levels of human NK cell cytotoxicity andNK-mediated antibody dependent cellular cytotoxicity (ADCC) againstpancreatic tumor cell targets induced by hIL7/IL21/TxM fusion proteincomplex; compared to ALT-803 or control media. Data from NK cellsisolated 2 different donors is shown.

FIG. 20 are bar graphs showing levels of IFNγ released by human NK cellsin response to hIL7/IL21/TxM fusion protein complex; compared to ALT-803or control media. Data from NK cells isolated 2 different donors isshown.

FIG. 21 is a bar graph showing levels of granzyme B expressed by humanNK cells in response to hIL7/IL21/TxM fusion protein complex; comparedto ALT-803 or control media. Data from NK cells isolated 2 differentdonors is shown.

FIGS. 22A-22E shows a series of graphs demonstrating the capture anddetection of IL-15, IL-7 and IL-21 components in hIL7/IL21/TxM.

FIGS. 23A-23C are a series of graphs showing that hIL7/IL21/TxM inducesspecific activation of IL-7 (FIG. 23A), IL-21 (FIG. 23B) and IL-15 (FIG.23C) receptors. FIG. 23A: IL-7 dependent 2E8 cells (10⁵) were stimulatedfor 2 days with hIL7/IL21/TxM or IL-7 and cell proliferation wasassessed using PrestoBlue. The EC₅₀ of IL-7 in hIL7/IL21/TxM is 14 pM.n=4 from 2 experiments. FIG. 23B: Activated natural killer (aNK) cellsaNK cells (2×10⁵) were stimulated for 40 hours with hIL7/IL21/TxM orN-803 and production of IFNγ was measured by ELISA. n=2 from 1experiment. FIG. 23C: IL-2/15 dependent 32D-IL2/15Rβ cells (10⁴) werestimulated for 3 days with hIL7/IL21/TxM or N-803 and cell proliferationwas assessed using PrestoBlue. The EC₅₀ of IL-15 in hIL7/IL21/TxM is 530pM. n=4 from 2 experiments.

FIG. 24 is a graph demonstrating the enhancement of granzyme Bexpression in human NK cells induced by hIL7/IL21/TxM. Granzyme Bexpression in pre-activated human NK cells (16 h, 50 nM, n=2).

FIGS. 25A, 25B are graphs demonstrating the cytotoxicity and ADCCactivity of hIL7/IL21/TxM-activated human NK cells against SW1990pancreatic cancer cells. Fresh NK cells were mixed with SW1990 cells for40 h at E:T of 2:1. αTF=0.1 nM. N-803 or hIL7/IL21/TxM=50. FIG. 25A: NKcell cytotoxicity. FIG. 25B: NK cell cytotoxicity-associated IFNγ.

FIG. 26 is a graph demonstrating the cytotoxicity and ADCC activity ofhIL7/IL21/TxM-activated human NK cells against Ramos lymphoma cells.Fresh NK cells were mixed with Ramos cells for 40 h at E:T of 1:1.αCD20=1 nM. N-803 or hIL7/IL21/TxM=0.5 nM. To show n=2.

FIG. 27A is a schematic representation of an embodiment of the methodused to demonstrate that hIL7/IL21/TxM is superior to individualcytokines in expanding purified NK cells. The results obtained are shownas a graph (FIG. 27B).

FIGS. 28A-28B is a series of graphs demonstrating that hIL7/IL21/TxM issuperior to individual cytokines in expanding NK cells from humandonors. (FIG. 28A—Donor 1; FIG. 28B—Donor 2.) FIGS. 29A-29C are a seriesof graphs comparing hIL7/IL21/TxM to other ex vivo NK expansion methods.FIG. 29A: Irradiated EBV lymphoblast feeder cells +IL-2 NK expansion.

FIG. 29B: K562-based activated antigen presenting cells (aAPCs) withmembrane-bound IL-21 (mbIL21). FIG. 29C: hIL7/IL21/TxM mediated NK cellexpansion.

FIGS. 30A-30D is a series of density plots showing the NK cell phenotypefollowing hIL7/IL21/TxM expansion.

FIGS. 31A-31C are graphs demonstrating that hIL7/IL21/TxM induces IFNγ(FIG. 31A), granzyme (FIG. 31B), and perforin (FIG. 31C) in NK cells.hIL7/IL21/TxM-expanded NK cells were stimulated by separate cytokines orTxM overnight.

FIG. 32 is a graph demonstrating the direct and antibody-mediatedcytotoxicity of hIL7/IL21/TxM-expanded NK cells: 9 days. Purified humanNK cells (0.5×10⁶/ml) were expanded with 20 nM hIL7/IL21/TxM for 9 d,washed once, and mixed with CellTrace Violet labeled CD20⁺ RamosBurkitt's lymphoma cells (10⁵) for 4 hr, followed by flow cytometryanalysis in the presence of 7-AAD viability reagent.

FIGS. 33A-33D depicts a series of graphs demonstrating the expansion ofsorted T cell populations following hIL7/IL21/TxM treatment. Sorted CD8⁺naïve (FIG. 33A), central memory (FIG. 33B), effector memory (FIG. 33C)and stem cell memory (FIG. 33D) T cells were labeled with CFSE andstimulated with media alone (US) or IL-7/IL-21 (25 ng), IL-7/N-803 (25ng/144 ng), IL-21/N-803 (25 ng/144 ng), IL-7/IL-21/N-803 (25 ng/25ng/144 ng), TxM (1.4 mg) in 200 ml total volume in 96 well flat bottomplate in 37° C., 5% CO₂.

FIGS. 34A-34D depicts a series of graphs demonstrating thathIL7/IL21/TxM effectively expands sorted CD8⁺ T cell populationsfollowing brief exposure to αCD3/CD28 beads. (FIG. 34A—Naïve; FIG.34B—Central Memory; FIG. 34C—Effector Memory; FIG. 34D—Stem CellMemory.)

FIG. 35 is a schematic representation of h2*IL21/TxM(IL15N72D:IL21-IL15RαSuFc).

FIGS. 36A and 36B are graphs demonstrating that h2*IL21/TxM inducesspecific activation of IL-21 (FIG. 36A) and IL-15 (FIG. 36B) receptors.FIG. 36A: aNK cells (2×10⁵) were stimulated for 40 hours withh2*IL21/TxM or N-803 and production of IFNγ was measured by ELISA. n=2from 1 experiment. FIG. 36B: IL-2/15 dependent 32D-IL2/15Rβ cells (10⁴)were stimulated for 3 days with h2*IL21/TxM or N-803 and cellproliferation was assessed using PrestoBlue. The EC₅₀ of IL-15 inh2*IL21/TxM is 56 pM. n=4 from 2 experiments.

FIG. 37 is a schematic representation of h2*IL7(IL15)/TxM(IL7-IL15N72D:IL15RαSuFc).

FIGS. 38A-38C are graphs showing that h2*IL7(IL15)/TxM induces specificactivation of IL-7 (FIG. 38A) and IL-15 (FIG. 38C) receptors. FIG. 38A:IL-7 dependent 2E8 cells (10⁵) were stimulated for 2 days withh2*IL7(IL15)/TxM or IL-7 and cell proliferation was assessed usingPrestoBlue. The EC₅₀ of IL-7 in h2*IL7(IL15)/TxM is 13.3 pM. n=4 from 2experiments.

FIG. 38B: aNK cells (2×10⁵) were stimulated for 40 hours withh2*IL7(IL15)/TxM or N-803 and production of IFNγ was measured by ELISA.n=2 from 1 experiment. FIG. 38C: IL-2/15 dependent 32D-IL2/15Rβ cells(10⁴) were stimulated for 3 days with h2*IL7(IL15)/TxM or N-803 and cellproliferation was assessed using PrestoBlue. The EC₅₀ of IL-15 inh2*IL7(IL15)/TxM is 81.3 pM. n=4 from 2 experiments.

FIG. 39 is a schematic representation showing a comparison of thestructures of hIL7/IL21/TxM vs. h2*IL21/TxM vs. h2*IL7(IL15)/TxM.

FIGS. 40A-40B show hIL7/IL21/TxM vs. h2*IL21/TxM vs. h2*IL7(IL15)/TxMrun on gels under reduced (FIG. 40A) and non-reduced (FIG. 40B)conditions.

FIG. 41 is a graph demonstrating NK cell expansion stimulated withhIL7/IL21/TxM vs. h2*IL21/TxM vs. h2*IL7(IL15)/TxM. Purified human NKcells were stimulated with 19.4 nM hIL7/IL21/TxM, h2*IL21/TxM, orhIL7(IL15)/TxM, and cell number was maintained between 0.5-2×10⁶/ml.Cell number assessed with Vi-CELL XR. n=2 from 1 experiment.

FIG. 42 is a graph showing enhanced antigen specific CD8 T cellresponses in the presence of superkine (hIL7IL15//IL21/TxM fusionprotein complex. T cells were selected from non-adherent PBMC, thencultured with a TCR stimulus (CD2/3/28 agonist Abs, 2 d), then washed ofTCR stimulus, followed by culture with IL-7/15/21 superkine. After Tcell expansion, T cells were added to autologous dendritic cells (DC),which were derived from the adherent fraction of PBMC. To the T/DCcultures were also added, nothing, BL21 E. coli expressing no pp65 (fromCMV) or BL21 expressing pp65. Cultures were stimulated overnight, thenELISPOT developed to indicate the number of spot forming cells (IFN-γproducing)

DETAILED DESCRIPTION

Therapies employing natural killer (NK) cells and T cells have emergedas potential treatments for cancer and viral infections due to theability of these cells to kill diseased cells and releasepro-inflammatory cytokines (See, e.g., Fehniger T A and Cooper M A.Trends Immunol. 2016; 37:877-888; and Cerwenka A and Lanier L L. Nat RevImmunol. 2016 16:112-23). Of particular interest is adoptive transfer ofT cells genetically engineered to express chimeric antigen receptors(CARs) for the induction of tumor-specific immune responses. The effectof cytokines on the phenotype of CAR T cells has been previouslydescribed. Stimulation with IL-2, IL-7, and IL-15 led to ex vivoexpansion of CAR T cells better than other cytokines or no cytokinepresence. (Nayar S., et al., OncoImmunology, 2014; 4:e1002720;Golubovskaya V. and Wu, L. Cancers 2016; 8:236; Sabatino M. et al.Blood. 2016; 128:519-528; Xu Y, et al. Blood. 2014:123:3750-3759; andGomez-Eerland R, et al. Hum Gene Ther Methods. 2014; 25:277-287).

Recent clinical data suggest that adoptive transfer ofless-differentiated T cells, in particular memory stem T cells(T_(SCM)), can trigger profound and durable tumor eradication (See, e.g.Klebanoff C A et al. PNAS. 2005; 102(27):9571-9576; and Sommermeyer D,et al. Leukemia. 2016:30(2):492-500). Due to the low numbers of T_(SCM)cells in circulation, it has been a challenge isolating and producingrelevant numbers of clinical grade T_(SCM) cells for adoptive celltherapy (See, e.g. Gattinoni L, et al. Blood. 2013; 121(4):567-568). Newreports have shown that generation and expansion of T_(SCM) cells couldbe achieved ex vivo using CD3/CD28 costimulation and the addition ofIL-7, IL-21, and IL-15 throughout the entire culture period (See, e.g.Alvarez-Fernabdez C, et al. J Trans Med. 2016; 14; 214; and Sabatino Met al. Blood. 2016; 128(4):519-528). IL-7 was shown to most effective atincreasing proliferation of CAR T_(SCM) cells, and IL-21 supported theexpansion of CAR T cells with more stem cell-like phenotype, while IL-2induced more differentiated CAR T cells. IL-2 and IL-15-treated CAR Tcells produced more pro-inflammatory cytokines and exhibited increasedantitumor activity in vitro. Additionally, treatment with IL-15 andIL-21 with CAR T cells in vivo increased their tumor cell lysis ability.

IL-7, IL-15, and IL-21, members of the four-helix common gamma chaincytokines, are pivotal in the differentiation, development, maturation,proliferation, and activation of Natural Killers (NK) cells (Waldman TNature Rev. Immunology 2006; 6:595-601; Leonard W. J. and Wan C.-K.F1000Research 2016; 5:244; Lin J. et al. Anticancer Research 2017;37:936-968). Adoptive transfer of NK cell is a promising immune therapyagainst cancer and infectious agents. The major challenge in NK celltherapy is the requirement of a large numbers of highly cytotoxic NKcells. Thus, ex vivo NK cell expansion approaches are being developedand majority of these culturing strategies are based on the use offeeder or accessory cells which need to be removed prior to the clinicalapplication of the final NK cell product (Tong A. A. et al.OncoImmunology 2017; 6:e1303586; Denman C J PLos One 7:e30264; FujisakiH. et al. Cancer Research 2009; 69:4010-4017). Recently, approaches ofusing common gamma chain cytokines, particularly IL-15 and IL-21, in theabsence of feeder cells for expansion and activation were explored(Wagner J. et al., Frontier in Immunology 2017; 8:676).

Prior to the invention described herein, optimal methods for generatingand expanding T_(SCM) cells were not fully elucidated. Strategiesemployed recombinant human IL-7, human IL-21, and human IL-15, whichdiffer in glycosylation and potentially other post-transcriptionalmodifications compared to mammalian cell-produced cytokines. Therecombinant cytokines may also have different purity and stability andare not generally available as clinical grade material. Additionally,each cytokine is expected to have unique receptor binding,internalization and recycling properties.

Accordingly, described herein are multi-specific IL-15-based proteincomplexes comprising IL-7 and IL-21 binding domains (FIGS. 1A, 1B).Specifically, described herein are protein complexes comprising anIL-15N72D:IL-15RαSu-Ig Fc scaffold fused to IL-7 and IL-21 bindingdomains. When characterized using human immune cells, these complexesexhibit binding and biological activity of each of the IL-15, IL-7 andIL-21 cytokines. Additionally, these complexes act to induceproliferation and activation of T cells with elevated T_(SCM) cellmarkers, and enhanced production of IFN-γ. These complexes also expandthe NK cells ex vivo and the expanded NK cells exhibit augmentedcytotoxicity. Thus, the complex as a single molecule binds to andsignals via multiple cytokine receptors on T and NK cells to provide thesynergistic responses previously only observed with a combination ofmultiple cytokines. Additionally, these complexes comprise the Fc regionof Ig molecules, which can form a dimer to provide a solublemulti-polypeptide complex, bind Protein A for the purpose ofpurification and interact with Fcγ receptors on NK cells and macrophagesfor transpresentation, thus providing advantages to the complex that arenot present in the combination of individual cytokines. Mammalian cellexpression-based methods produce these complexes as glycosylatedproteins which may have better activity and/or stability. These methodsare also suitable for production of clinical grade material as describedherein. Additional methods for inducing proliferation and activation ofNK and T cells and generating T_(SCM) cells and CIML NK cells induced bythe protein complex of the invention are also provided.

Interleukin-15

Interleukin-15 (IL-15) is an important cytokine for the development,proliferation, and activation of effector NK cells and CD8⁺ memory Tcells. IL-15 binds to the IL-15 receptor α (IL-15Rα) and is presented intrans to the IL-2/IL-15 receptor β-common γ chain (IL-15Rβγ_(c)) complexon effector cells. IL-15 and IL-2 share binding to the IL-15Rβγ_(c), andsignal through STAT3 and STATS pathways. However, unlike IL-2, IL-15does not support maintenance of CD4⁺CD25⁺FoxP3⁺ regulatory T (Treg)cells or induce cell death of activated CD8⁺ T cells, effects that mayhave limited the therapeutic activity of IL-2 against multiple myeloma.Additionally, IL-15 is the only cytokine known to provide anti-apoptoticsignaling to effector CD8⁺ T cells. IL-15, either administered alone oras a complex with the IL-15Rα, exhibits potent anti-tumor activitiesagainst well-established solid tumors in experimental animal models and,thus, has been identified as one of the most promising immunotherapeuticdrugs that could potentially cure cancer.

To facilitate clinical development of an IL-15-based cancer therapeutic,an IL-15 mutant (IL-15N72D) with increased biological activity comparedto IL-15 was identified (Zhu et al., J Immunol, 183: 3598-3607, 2009).The pharmacokinetics and biological activity of this IL-15 super-agonist(IL-15N72D) was further improved by the creation of IL-15N72D:IL-15Rα/Fcfusion complex (ALT-803), such that the super agonist complex has atleast 25-times the activity of the native cytokine in vivo (Han et al.,Cytokine, 56: 804-810, 2011).

IL-15:IL-15Rα Complex

As described above, an IL-15:IL-15Rα fusion protein complex can refer toa complex having IL-15 non-covalently bound to the soluble IL-15Rαdomain of the native IL-15Rα. In some cases, the soluble IL-15Rα iscovalently linked to a biologically active polypeptide and/or to an IgGFc domain. The IL-15 can be either IL-15 or IL-15 covalently linked to asecond biologically active polypeptide. The crystal structure of theIL-15:IL-15Rα complex is shown in Chirifu et al., 2007 Nat Immunol 8,1001-1007, incorporated herein by reference.

In various embodiments of the above aspects or any other aspect of theinvention delineated herein, the IL-15Rα fusion protein comprisessoluble IL-15Rα, e.g., IL-15Rα covalently linked to a biologicallyactive polypeptide (e.g., the heavy chain constant domain of IgG, an Fcdomain of the heavy chain constant domain of IgG, or a cytokine). Inother embodiments of the invention of the above aspects, IL-15 comprisesIL-15, e.g., IL-15 covalently linked to a second biologically activepolypeptide, e.g., a cytokine. In other embodiments, purifying theIL-15:IL-15Rα complex from the host cell or media involves capturing theIL-15:IL-15Rα complex on an affinity reagent that specifically binds theIL-15:IL-15Rα fusion protein complex. In other embodiments, the IL-15Rαfusion protein contains an IL-15Rα/Fc fusion protein and the affinityreagent specifically binds the Fc domain. In other embodiments, theaffinity reagent is Protein A or Protein G. In other embodiments, theaffinity reagent is an antibody. In other embodiments, purifying theIL-15:IL-15Rα complex from the host cell or media comprises ion exchangechromatography. In other embodiments, purifying the IL-15:IL-15Rαcomplex from the host cell or media comprises size exclusionchromatography.

In other embodiments, the IL-15Rα comprises IL-15RαSushi (IL-15RαSu). Inother embodiments, the IL-15 is a variant IL-15 (e.g., IL-15N72D). Inother embodiments, the IL-15 binding sites of the IL-15:IL-15Rα complexare fully occupied. In other embodiments, both IL-15 binding sites ofthe IL-15:IL-15RαSu/Fc complex are fully occupied. In other embodiments,the IL-15:IL-15Rα complex is purified based on the complex charge orsize properties. In other embodiments, the fully occupiedIL-15N72D:IL-15RαSu/Fc fusion protein complex is purified by anionexchange chromatography based on the complex charge properties. In otherembodiments, the fully occupied IL-15N72D:IL-15RαSu/Fc fusion proteincomplex is purified using a quaternary amine-based resin with bindingconditions employing low ionic strength neutral pH buffers and elutionconditions employing buffers of increasing ionic strength.

In certain embodiments of the soluble fusion protein complexes of theinvention, the IL-15 polypeptide is an IL-15 variant having a differentamino acid sequence than native IL-15 polypeptide. The human IL-15polypeptide is referred to herein as huIL-15, hIL-15, huIL15, hIL15,IL-15 wild type (wt) and variants thereof are referred to using thenative amino acid, its position in the mature sequence and the variantamino acid. For example, huIL15N72D refers to human IL-15 comprising asubstitution of N to D at position 72. In certain embodiments, the IL-15variant functions as an IL-15 agonist as demonstrated, e.g., byincreased binding activity for the IL-15RβγC receptors compared to thenative IL-15 polypeptide. In certain embodiments, the IL-15 variantfunctions as an IL-15 antagonist as demonstrated by e.g., decreasedbinding activity for the IL-15RβγC receptors compared to the nativeIL-15 polypeptide. In certain embodiments, the IL-15 variant hasincreased binding affinity or a decreased binding activity for theIL-15RβγC receptors compared to the native IL-15 polypeptide. In certainembodiments, the sequence of the IL-15 variant has at least one (i.e.,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid change compared tothe native IL-15 sequence. The amino acid change can include one or moreof an amino acid substitution or deletion in the domain of IL-15 thatinteracts with IL-15Rβ and/or IL-15RγC. In certain embodiments, theamino acid change is one or more amino acid substitutions or deletionsat position 8, 61, 65, 72, 92, 101, 108, or 111 of the mature humanIL-15 sequence. For example, the amino acid change is the substitutionof D to N or A at position 8, D to A at position 61, N to A at position65, N to R at position 72 or Q to A at position 108 of the mature humanIL-15 sequence, or any combination of these substitutions. In certainembodiments, the amino acid change is the substitution of N to D atposition 72 of the mature human IL-15 sequence.

ALT-803 (N-803)

ALT-803 comprises an IL-15 mutant with increased ability to bind IL-2Rβγand enhanced biological activity (U.S. Pat. No. 8,507,222, incorporatedherein by reference). This super-agonist mutant of IL-15 was describedin a publication (Zu et al., 2009 J Immunol, 183: 3598-3607,incorporated herein by reference). This IL-15 super-agonist incombination with a soluble IL-15a receptor fusion protein (IL-15RαSu/Fc)results in a protein complex with highly potent IL-15 activity in vitroand in vivo (Han et al., 2011, Cytokine, 56: 804-810; Xu, et al., 2013Cancer Res. 73:3075-86, Wong, et al., 2013, OncoImmunology 2:e26442).The IL-15 super agonist complex (IL-15N72D:IL-15RαSu/Fc) is referred toas “ALT-803.”

Pharmacokinetic analysis indicated that the complex has a half-life of25 hours following i.v. administration in mice. ALT-803 exhibitsimpressive anti-tumor activity against aggressive solid andhematological tumor models in immunocompetent mice. It can beadministered as a monotherapy using a twice weekly or weekly i.v. doseregimen or as combinatorial therapy with an antibody. The ALT-803anti-tumor response is also durable. Tumor-bearing mice that were curedafter ALT-803 treatment were also highly resistant to re-challenge withthe same tumor cells indicating that ALT-803 induces effectiveimmunological memory responses against the re-introduced tumor cells.

IL-7

IL-7 is a cytokine essential for adaptive immune cells development,survival and proliferation. While IL-7 is secreted mainly by stromalcells in the bone marrow and thymus, other immune cells, such asdendritic cells (DCs) can also produce IL-7. The IL-7 receptor is aheterodimer consisting of two chains: IL-7Rα (CD127), which is sharedwith thymic stromal lymphopoietin (TSLP), and the common γ chain (CD132)which is shared with IL-2, IL-4, IL-9, IL-15 and IL-21. The γ chain isexpressed on all hematopoietic cell types, while IL-7Rα is expressedmostly on lymphocytes. IL-7Rα is also found in innate lymphoid cells(ILCs), such as NK cells and gut-associated lymphoid tissue(GALT)-derived LTi cells which are critical in lymphoid organdevelopment and innate immune responses to pathogens. IL-7 can alsoregulate lymphoid organogenesis by controlling the pool of LTi cells.Another type of IL-7 receptor is a soluble IL-7R, which competes withcell-associated IL-7R to reduce excessive IL-7 consumption byIL-7R-expressing cells and enhances the bioactivity of IL-7 when thecytokine is limited (Gao J et al. Int J Mol Sci. 2015; 16(10267-10280)).

Two main signaling pathways are responsible for the action of IL-7:Jak-Stat and PI3K-Akt. IL-7Rα is associated with the protein tyrosinekinase Janus kinase 1 (Jak1), and the cytosolic tail of the γ chain isassociated with Jak3. Binding of IL-7 to its receptor leads to theactivation of Jak in the cytosol, phosphorylating Stat proteins. Thedimeric phosphorylated Stat proteins subsequently translocate into thenucleus and induce gene expression. Via the Jak3-Stat5 pathway, IL-7activates the anti-apoptotic genes, Bcl-2 and Mcl-1, and suppressespro-apoptotic proteins, such as Bax and Bak, which in turn leads tonaïve and memory T cells survival. This function is dose-dependent, suchthat a higher concentration of IL-7 induces thymic emigrant T cellproliferation, while lower concentrations sustain cell survival. Byactivating the PI3K-Akt pathway, IL-7 downregulates the cell cycleinhibitor p27kip1 to induce the expression of cyclin D1 for cell cycleprogression. Moreover, it promotes glucose transporter 1 expression,glucose uptake and mitochondrial integrity to positively regulate cellmetabolism and size (Gao J et al. Int J Mol Sci. 2015; 16(10267-10280);and Jatiani S S et al. Genes Cancer. 2010; 1(10):979-993).

IL-21

IL-21 is a pleiotropic cytokine that has both pro- and anti-inflammatoryactivities and is mainly produced by activated CD4⁺ and NK T cells. Itbelongs to the common γ-chain cytokine family and is involved inlymphocyte activation, proliferation, differentiation, and survival.IL-21 functions via heterodimer receptor signaling consisting ofspecific IL-21R and the common γ-chain receptor. IL-21 signals throughthe Jak-Stat, PI3K, and MAPK pathways. IL-21 induces strong andcontinued activation of Stat3, which is critical for T-celldifferentiation (Ouyang W, et al. Immunity. 2012; 28(4)454-467).

Together with IL-15 and IL-7, IL-21 promotes expansion ofantigen-specific CD8⁺ T-cell numbers and their effector function,resulting in tumor regression. IL-21 has also been shown to play animportant role in the development and survival of both naïve and centralmemory T cells by the induction of an early differentiation phenotype.Importantly, T cells generated with IL-21 showed a superior antitumoreffect in vivo in experimental models.

IL-21 has also been shown to synergize with IL-2, IL-15, and Flt-3L ingenerating NK cells. It was recently reported that while IL-15 has arole in expanding NK cells, IL-21 induces cytotoxic activity byincreased degranulation and secretion of inflammatory cytokines (WagnerJ et al. Front Immunol. 2017; 8:676).

Antigen-Specific Binding Domains

Antigen-specific binding domains consist of polypeptides thatspecifically bind to targets on diseased cells. Alternatively, thesedomains may bind to targets on other cells that support the diseasedstate, such as targets on stromal cells that support tumor growth ortargets on immune cells that support disease-mediated immunosuppression.Antigen-specific binding domains include antibodies, single chainantibodies, Fabs, Fv, T-cell receptor binding domains, ligand bindingdomains, receptor binding domains, domain antibodies, single domainantibodies, minibodies, nanobodies, peptibodies, or various otherantibody mimics (such as affimers, affitins, alphabodies, atrimers,CTLA4-based molecules, adnectins, anticalins, Kunitz domain basedproteins, avimers, knottins, fynomers, darpins, affibodies, affilins,monobodies and armadillo repeat protein-based proteins (Weidle, U H, etal. 2013. Cancer Genomics & Proteomics 10: 155-168)) known in the art.

In certain embodiments, the antigen for the antigen-specific bindingdomain comprises a cell surface receptor or ligand. In a furtherembodiment, the antigen comprises a CD antigen, cytokine or chemokinereceptor or ligand, growth factor receptor or ligand, tissue factor,cell adhesion molecule, MHC/MHC-like molecules, Fc receptor, Toll-likereceptor, NK receptor, TCR, BCR, positive/negative co-stimulatoryreceptor or ligand, death receptor or ligand, tumor associated antigen,or virus encoded antigen.

Preferably, the antigen-specific binding domain is capable of binding toan antigen on a tumor cell. Tumor-specific binding domain may be derivedfrom antibodies approved for treatment of patients with cancer includerituximab, ofatumumab, and obinutuzumab (anti-CD20 Abs); trastuzumab andpertuzumab (anti-HER2 Abs); cetuximab and panitumumab (anti-EGFR Abs);and alemtuzumab (anti-CD52 Ab). Similarly, binding domains from approvedantibody effector molecule conjugates specific to CD20 (⁹⁰Y-labeledibritumomab tiuxetan, ¹³¹I-labeled tositumomab), HER2 (ado-trastuzumabemtansine), CD30 (brentuximab vedotin) and CD33 (gemtuzumab ozogamicin)(Sliwkowski M X, Mellman I. 2013 Science 341:1192) could be used.

Additionally, preferred binding domains of the invention may includevarious other tumor-specific antibody domains known in the art. Theantibodies and their respective targets for treatment of cancer includebut are not limited to nivolumab (anti-PD-1 Ab), TA99 (anti-gp75), 3F8(anti-GD2), 8H9 (anti-B7-H3), abagovomab (anti-CA-125 (imitation)),adecatumumab (anti-EpCAM), afutuzumab (anti-CD20), alacizumab pegol(anti-VEGFR2), altumomab pentetate (anti-CEA), amatuximab(anti-mesothelin), AME-133 (anti-CD20), anatumomab mafenatox(anti-TAG-72), apolizumab (anti-HLA-DR), arcitumomab (anti-CEA),bavituximab (anti-phosphatidylserine), bectumomab (anti-CD22), belimumab(anti-BAFF), besilesomab (anti-CEA-related antigen), bevacizumab(anti-VEGF-A), bivatuzumab mertansine (anti-CD44 v6), blinatumomab(anti-CD19), BMS-663513 (anti-CD137), brentuximab vedotin (anti-CD30(TNFRSF8)), cantuzumab mertansine (anti-mucin CanAg), cantuzumabravtansine (anti-MUC1), capromab pendetide (anti-prostatic carcinomacells), carlumab (anti-MCP-1), catumaxomab (anti-EpCAM, CD3),cBR96-doxorubicin immunoconjugate (anti-Lewis-Y antigen), CC49(anti-TAG-72), cedelizumab (anti-CD4), Ch. 14.18 (anti-GD2), ch-TNT(anti-DNA associated antigens), citatuzumab bogatox (anti-EpCAM),cixutumumab (anti-IGF-1 receptor), ivatuzumab tetraxetan (anti-MUC1),conatumumab (anti-TRAIL-R2), CP-870893 (anti-CD40), dacetuzumab(anti-CD40), daclizumab (anti-CD25), dalotuzumab (anti-insulin-likegrowth factor I receptor), daratumumab (anti-CD38 (cyclic ADP ribosehydrolase)), demcizumab (anti-DLL4), detumomab (anti-B-lymphoma cell),drozitumab (anti-DR5), duligotumab (anti-HERβ), dusigitumab(anti-ILGF2), ecromeximab (anti-GD3 ganglioside), edrecolomab(anti-EpCAM), elotuzumab (anti-SLAMF7), elsilimomab (anti-IL-6),enavatuzumab (anti-TWEAK receptor), enoticumab (anti-DLL4), ensituximab(anti-5AC), epitumomab cituxetan (anti-episialin), epratuzumab(anti-CD22), ertumaxomab (anti-HER2/neu, CD3), etaracizumab(antiintegrin av-3), faralimomab (anti-Interferon receptor),farletuzumab (anti-folate receptor 1), FBTAO5 (anti-CD20), ficlatuzumab(anti-HGF), figitumumab (anti-IGF-1 receptor), flanvotumab (anti-TYRP1(glycoprotein 75)), fresolimumab (anti-TGF ˜), futuximab (anti-EGFR),galiximab (anti-CD80), ganitumab (anti-IGF-1), gemtuzumab ozogamicin(anti-CD33), girentuximab (anti-carbonic anhydrase 9 (CA-IX)),glembatumumab vedotin (anti-GPNMB), guselkumab (anti-IL13), ibalizumab(anti-CD4), ibritumomab tiuxetan (anti-CD20), icrucumab (anti-VEGFR-1),igovomab (anti-CA-125), IMAB362 (anti-CLDN18.2), IMC-CS4 (anti-CSF1R),IMC-TR1 (TGF-RII), imgatuzumab (anti-EGFR), inclacumab (anti-selectinP), indatuximab ravtansine (anti-SDC1), inotuzumab ozogamicin(anti-CD22), intetumumab antiCD51), ipilimumab (anti-CD152), iratumumab(anti-CD30 (TNFRSF8)), KM3065 (anti-CD20), KW-0761 (anti-CD194),LY2875358 (anti-MET) labetuzumab (anti-CEA), lambrolizumab (antiPDCDl),lexatumumab (anti-TRAIL-R2), lintuzumab (anti-CD33), lirilumab(anti-KIR2D), lorvotuzumab mertansine (anti-CD56), lucatumumab(anti-CD40), lumiliximab (anti-CD23 (IgE receptor)), mapatumumab(anti-TRAIL-R1), margetuximab (anti-ch4D5), matuzumab (anti-EGFR),mavrilimumab (anti-GMCSF receptor a-chain), milatuzumab (anti-CD74),minretumomab (anti-TAG-72), mitumomab (anti-GD3 ganglioside),mogamulizumab (antiCCR4), moxetumomab pasudotox (anti-CD22), nacolomabtafenatox (anti-C242 antigen), naptumomab estafenatox (anti-5T4),narnatumab (anti-RON), necitumumab (anti-EGFR), nesvacumab(anti-angiopoietin 2), nimotuzumab (anti-EGFR), nivolumab (anti-IgG4),nofetumomab merpentan, ocrelizumab (anti-CD20), ocaratuzumab(anti-CD20), olaratumab (anti-PDGF-R a), onartuzumab (anti-c-MET),ontuxizumab (anti-TEM1), oportuzumab monatox (anti-EpCAM), oregovomab(anti-CA-125), otlertuzumab (anti-CD37), pankomab (anti-tumor specificglycosylation of MU Cl), parsatuzumab (anti-EGFL7), pascolizumab(anti-IL-4), patritumab (anti-HERβ), pemtumomab (anti-MUC1), pertuzumab(anti-HER2/neu), pidilizumab (anti-PD-1), pinatuzumab vedotin(anti-CD22), pintumomab (anti-adenocarcinoma antigen), polatuzumabvedotin (anti-CD79B), pritumumab (anti-vimentin), PRO131921 (anti-CD20),quilizumab (anti-IGHE), racotumomab (anti-N-glycolylneuraminic acid),radretumab (anti-fibronectin extra domain-B), ramucirumab (anti-VEGFR2),rilotumumab (anti-HGF), robatumumab (anti-IGF-1 receptor), roledumab(anti-RHD), rovelizumab (anti-CD11 & CD18), samalizumab (anti-CD200),satumomab pendetide (anti-TAG-72), seribantumab (anti-ERBB3), SGN-CD19A(anti-CD19), SGN-CD33A (anti-CD33), sibrotuzumab (anti-PAP), siltuximab(anti-IL-6), solitomab (anti-EpCAM), sontuzumab (anti-episialin),tabalumab (anti-BAFF), tacatuzumab tetraxetan (anti-alpha-fetoprotein),taplitumomab paptox (anti-CD19), telimomab aritox, tenatumomab(anti-tenascin C), teneliximab (anti-CD40), teprotumumab (anti-CD221),TGN1412 (anti-CD28), ticilimumab (anti-CTLA-4), tigatuzumab(anti-TRAIL-R2), TNX-650 (anti-IL-13), tositumomab (anti-CS20),tovetumab (anti-CD140a), TRBS07 (anti-GD2), tregalizumab (anti-CD4),tremelimumab (anti-CTLA-4), TRU-016 (anti-CD37), tucotuzumab celmoleukin(anti-EpCAM), ublituximab (anti-CD20), urelumab (anti-4-1BB),vantictumab (anti-Frizzled receptor), vapaliximab (anti-AOC3 (VAP-1)),vatelizumab (anti-ITGA2), veltuzumab (anti-CD20), vesencumab(anti-NRP1), visilizumab (anti-CD3), volociximab (antiintegrin a5(31),vorsetuzumab mafodotin (anti-CD70), votumumab (anti-tumor antigenCTAA16.88), zalutumumab (anti-EGFR), zanolimumab (anti-CD4), zatuximab(anti-HER1), ziralimumab (anti-CD147 (basigin)), RG7636 (anti-ETBR),RG7458 (anti-MUC16), RG7599 (anti-NaPi2b), MPDL3280A (anti-PD-L1),RG7450 (anti-STEAP1), and GDC-0199 (anti-Bcl-2).

Other antibody domains or tumor target binding proteins useful in theinvention (e.g. TCR domains) include, but are not limited to, those thatbind the following antigens (note, the cancer indications indicatedrepresent non-limiting examples): aminopeptidase N (CD13), annexin A1,B7-H3 (CD276, various cancers), CA125 (ovarian cancers), CA15-3(carcinomas), CA19-9 (carcinomas), L6 (carcinomas), Lewis Y(carcinomas), Lewis X (carcinomas), alpha fetoprotein (carcinomas),CA242 (colorectal cancers), placental alkaline phosphatase (carcinomas),prostate specific antigen (prostate), pro static acid phosphatase(prostate), epidermal growth factor (carcinomas), CD2 (Hodgkin'sdisease, NHL lymphoma, multiple myeloma), CD3 epsilon (T cell lymphoma,lung, breast, gastric, ovarian cancers, autoimmune diseases, malignantascites), CD19 (B cell malignancies), CD20 (non-Hodgkin's lymphoma, Bcell neoplasmas, autoimmune diseases), CD21 (B-cell lymphoma), CD22(leukemia, lymphoma, multiple myeloma, SLE), CD30 (Hodgkin's lymphoma),CD33 (leukemia, autoimmune diseases), CD38 (multiple myeloma), CD40(lymphoma, multiple myeloma, leukemia (CLL)), CD51 (metastatic melanoma,sarcoma), CD52 (leukemia), CD56 (small cell lung cancers, ovariancancer, Merkel cell carcinoma, and the liquid tumor, multiple myeloma),CD66e (carcinomas), CD70 (metastatic renal cell carcinoma andnon-Hodgkin lymphoma), CD74 (multiple myeloma), CD80 (lymphoma), CD98(carcinomas), CD123 (leukemia), mucin (carcinomas), CD221 (solidtumors), CD227 (breast, ovarian cancers), CD262 (NSCLC and othercancers), CD309 (ovarian cancers), CD326 (solid tumors), CEACAM3(colorectal, gastric cancers), CEACAM5 (CEA, CD66e) (breast, colorectaland lung cancers), DLL4 (A-like-4), EGFR (various cancers), CTLA4(melanoma), CXCR4 (CD184, heme-oncology, solid tumors), Endoglin (CD105,solid tumors), EPCAM (epithelial cell adhesion molecule, bladder, head,neck, colon, NHL prostate, and ovarian cancers), ERBB2 (lung, breast,prostate cancers), FCGR1 (autoimmune diseases), FOLR (folate receptor,ovarian cancers), FGFR (carcinomas), GD2 ganglioside (carcinomas), G-28(a cell surface antigen glycolipid, melanoma), GD3 idiotype(carcinomas), heat shock proteins (carcinomas), HER1 (lung, stomachcancers), HER2 (breast, lung and ovarian cancers), HLA-DR1O (NHL),HLA-DRB (NHL, B cell leukemia), human chorionic gonadotropin(carcinomas), IGF1R (solid tumors, blood cancers), IL-2 receptor (T-cellleukemia and lymphomas), IL-6R (multiple myeloma, RA, Castleman'sdisease, IL6 dependent tumors), integrins (αvβ3, αpβ51, α6β4, α11β3,α5β5, αvβ5, for various cancers), MAGE-1 (carcinomas), MAGE-2(carcinomas), MAGE-3 (carcinomas), MAGE 4 (carcinomas), anti-transferrinreceptor (carcinomas), p97 (melanoma), MS4A1 (membrane-spanning4-domains subfamily A member 1, Non-Hodgkin's B cell lymphoma,leukemia), MUC1 (breast, ovarian, cervix, bronchus and gastrointestinalcancer), MUC16 (CA125) (ovarian cancers), CEA (colorectal cancer), gp100(melanoma), MARTI (melanoma), MPG (melanoma), MS4A1 (membrane-spanning4-domains subfamily A, small cell lung cancers, NHL), nucleolin, Neuoncogene product (carcinomas), P21 (carcinomas), nectin-4 (carcinomas),paratope of anti-(N-glycolylneuraminic acid, breast, melanoma cancers),PLAP-like testicular alkaline phosphatase (ovarian, testicular cancers),PSMA (prostate tumors), PSA (prostate), ROB04, TAG 72 (tumor associatedglycoprotein 72, AML, gastric, colorectal, ovarian cancers), T celltransmembrane protein (cancers), Tie (CD202b), tissue factor, TNFRSF10B(tumor necrosis factor receptor superfamily member 10B, carcinomas),TNFRSF13B (tumor necrosis factor receptor superfamily member 13B,multiple myeloma, NHL, other cancers, RA and SLE), TPBG (trophoblastglycoprotein, renal cell carcinoma), TRAIL-R1 (tumor necrosis apoptosisinducing ligand receptor 1, lymphoma, NHL, colorectal, lung cancers),VCAM-1 (CD106, Melanoma), VEGF, VEGF-A, VEGF-2 (CD309) (variouscancers). Some other tumor associated antigen targets have been reviewed(Gerber, et al, mAbs 2009 1:247-253; Novellino et al, Cancer ImmunolImmunother. 2005 54:187-207, Franke, et al, Cancer Biother Radiopharm.2000, 15:459-76, Guo, et al., Adv Cancer Res. 2013; 119: 421-475,Parmiani et al. J Immunol. 2007 178: 1975-9). Examples of these antigensinclude Cluster of Differentiations (CD4, CDS, CD6, CD7, CDS, CD9, CD1O,CDI 1a, CDI 1b, CDI 1e, CD12w, CD14, CD15, CD16, CDwl7, CD18, CD21,CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD31, CD32, CD34, CD35, CD36,CD37, CD41, CD42, CD43, CD44, CD45, CD46, CD47, CD48, CD49b, CD49c,CD53, CD54, CD55, CD58, CD59, CD61, CD62E, CD62L, CD62P, CD63, CD68,CD69, CD71, CD72, CD79, CD81, CD82, CD83, CD86, CD87, CD88, CD89, CD90,CD91, CD95, CD96, CD100, CD103, CD105, CD106, CD109, CD117, CD120,CD127, CD133, CD134, CD135, CD138, CD141, CD142, CD143, CD144, CD147,CD151, CD152, CD154, CD156, CD158, CD163, CD166, CD168, CD184, CDw186,CD195, CD202 (a, b), CD209, CD235a, CD271, CD303, CD304), annexin A1,nucleolin, endoglin (CD105), ROB04, amino-peptidase N, -like-4 (DLL4),VEGFR-2 (CD309), CXCR4 (CD184), Tie2, B7-H3, WT1, MUC1, LMP2, HPV E6 E7,EGFRvlll, HER-2/neu, idiotype, MAGE A3, p53 nonmutant, NY-ESO-1, GD2,CEA, MelanA/MART1, Ras mutant, gp100, p53 mutant, proteinase3 (PR1),bcr-abl, tyrosinase, survivin, hTERT, sarcoma translocation breakpoints,EphA2, PAP, ML-IAP, AFP, EpCAM, ERG (TMPRSS2 ETS fusion gene), NA17,PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC,TRP-2, GD3, fucosyl GMI, mesothelin, PSCA, MAGE A1, sLe(a), CYPIB I,PLACI, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn,carbonic anhydrase IX, PAX5, OY-TES 1, sperm protein 17, LCK, HMWMAA,AKAP-4, SSX2, XAGE 1, B7H3, legumain, Tie 2, Page4, VEGFR2, MAD-CT-1,FAP, PDGFR-13, MAD-CT-2, and Fas-related antigen 1.

Additionally, preferred binding domains of the invention include thosespecific to antigens and epitope targets associated with infected cellsthat are known in the art. Such targets include but are not limitedthose derived from the following infectious agents are of interest: HIVvirus (particularly antigens derived from the HIV envelope spike and/orgp120 and gp41 epitopes), Human papilloma virus (HPV), Mycobacteriumtuberculosis, Streptococcus agalactiae, methicillin-resistantStaphylococcus aureus, Legionella pneumophilia, Streptococcus pyogenes,Escherichia coli, Neisseria gonorrhoeae, Neisseria meningitidis,Pneumococcus, Cryptococcus neoformans, Histoplasma capsulatum,-influenzae B, Treponema pallidum, Lyme disease spirochetes, Pseudomonasaeruginosa, Mycobacterium leprae, Brucella abortus, rabies virus,influenza virus, cytomegalovirus, herpes simplex virus I, herpes simplexvirus II, human serum parvo-like virus, respiratory syncytial virus,varicella-zoster virus, hepatitis B virus, hepatitis C virus, measlesvirus, adenovirus, human T-cell leukemia viruses, Epstein-Barr virus,murine leukemia virus, mumps virus, vesicular stomatitis virus, sindbisvirus, lymphocyticchoriomeningitis virus, wart virus, blue tongue virus,Sendai virus, feline leukemia virus, reovirus, polio virus, simian virus40, mouse mammary tumor virus, dengue virus, rubella virus, West Nilevirus, Plasmodium falciparum, Plasmodium vivax, Toxoplasma gondii,Trypanosoma rangeli, Trypanosoma cruzi, Trypanosoma rhodesiensei,Trypanosoma brucei, Schistosoma mansoni, Schistosoma japonicum, Babesiabovis, Elmeria tenella, Onchocerca volvulus, Leishmania tropica,Trichinella spiralis, Theileria parva, Taenia hydatigena, Taenia ovis,Taenia saginata, Echinococcus granulosus, Mesocestoides corti,Mycoplasma arthritidis, M. hyorhinis, M. orale, M arginini, Acholeplasmalaidlawii, M. salivarium and M. pneumoniae.

Immune Checkpoint Inhibitor and Immune Agonist Domains

In other embodiments, the binding domain is specific to an immunecheckpoint or signaling molecule or its ligand and acts as an inhibitorof immune checkpoint suppressive activity or as an agonist of immunestimulatory activity. Such immune checkpoint and signaling molecules andligands include PD-1, PD-L1 PD-L2, CTLA-4, CD28, CD80, CD86, B7-H3,B7-H4, B7-H5, ICOS-L, ICOS, BTLA, CD137L, CD137, HVEM, KIR, 4-1BB,OX40L, CD70, CD27, CD47, CIS, OX40, GITR, IDO, TIM3, GAL9, VISTA, CD155,TIGIT, LIGHT, LAIR-1, Siglecs and A2aR (Pardall D M. 2012. Nature RevCancer 12:252-264, Thaventhiran T, et al. 2012. J Clin Cell Immunol S12:004). Additionally, preferred antibody domains of the invention mayinclude ipilimumab and/or tremelimumab (anti-CTLA4), nivolumab,pembrolizumab, pidilizumab, TSR-042, ANBO11, AMP-514 and AMP-224 (aligand-Fe fusion) (anti-PD1), atezolizumab (MPDL3280A), avelumab(MSB0010718C), durvalumab (MEDI4736), MEDI0680, and BMS-9365569(anti-PDL1), MEDI6469 (anti-OX40 agonist), BMS-986016, IMP701, IMP731,IMP321 (anti-LAG3) and GITR ligand.

T-Cell Receptors (TCRs)

T cells are a subgroup of cells which together with other immune celltypes (polymorphonuclear cells, eosinophils, basophils, mast cells,B-cells, NK cells), constitute the cellular component of the immunesystem. Under physiological conditions, T cells function in immunesurveillance and in the elimination of foreign antigen. However, underpathological conditions, there is compelling evidence that T cells playa major role in the causation and propagation of disease. In thesedisorders, breakdown of T-cell immunological tolerance, either centralor peripheral is a fundamental process in the causation of autoimmunedisease.

The TCR complex is composed of at least seven transmembrane proteins.The disulfide-linked (αβ or γδ) heterodimer forms the monotypic antigenrecognition unit, while the invariant chains of CD3, consisting of ε, γ,δ, ζ, and η chains, are responsible for coupling the ligand binding tosignaling pathways that result in T-cell activation and the elaborationof the cellular immune responses. Despite the gene diversity of the TCRchains, two structural features are common to all known subunits. First,they are transmembrane proteins with a single transmembrane spanningdomain—presumably alpha-helical. Second, all TCR chains have the unusualfeature of possessing a charged amino acid within the predictedtransmembrane domain. The invariant chains have a single negativecharge, conserved between the mouse and human, and the variant chainspossess one (TCR-β) or two (TCR-α) positive charges. The transmembranesequence of TCR-α is highly conserved in a number of species and thusphylogenetically may serve an important functional role. The octapeptidesequence containing the hydrophilic amino acids arginine and lysine isidentical between the species.

A T-cell response is modulated by antigen binding to a TCR. One type ofTCR is a membrane bound heterodimer consisting of an α and β chainresembling an immunoglobulin variable (V) and constant (C) region. TheTCR α chain includes a covalently linked V-α and C-α chain, whereas theβ chain includes a V-β chain covalently linked to a C-β chain. The V-αand V-β chains form a pocket or cleft that can bind a superantigen orantigen in the context of a major histocompatibility complex (MHC)(known in humans as an HLA complex). See, Davis Ann. Rev. of Immunology3: 537 (1985); Fundamental Immunology 3rd Ed., W. Paul Ed. Rsen PressLTD. New York (1993).

The extracellular domains of the TCR chains (αβ or γδ) can alsoengineered as fusions to heterologous transmembrane domains forexpression on the cell surface. Such TCRs may include fusions to CD3,CD28, CD8, 4-1BB and/or chimeric activation receptor (CAR) transmembraneor activation domains. TCRs can also be the soluble proteins comprisingone or more of the antigen binding domains of αβ or γδ chains. Such TCRsmay include the TCR variable domains or function fragments thereof withor without the TCR constant domains. Soluble TCRs may be heterodimericor single-chain molecules.

Fc Domain

Protein complexes of the invention may contain an Fc domain. Forexample, hIL7/IL21/TxM comprises anIL-7/IL-15N72D:IL-21/IL-15RαSu/huIgG1 Fc fusion complex. Fusion proteinsthat combine the Fc regions of IgG with the domains of another protein,such as various cytokines and soluble receptors have been reported (see,for example, Capon et al., Nature, 337:525-531, 1989; Chamow et al.,Trends Biotechnol., 14:52-60, 1996); U.S. Pat. Nos. 5,116,964 and5,541,087). The prototype fusion protein is a homodimeric protein linkedthrough cysteine residues in the hinge region of IgG Fc, resulting in amolecule similar to an IgG molecule without the heavy chain variable andC_(H)1 domains and light chains. The dimeric nature of fusion proteinscomprising the Fc domain may be advantageous in providing higher orderinteractions (i.e. bivalent or bispecific binding) with other molecules.Due to the structural homology, Fc fusion proteins exhibit an in vivopharmacokinetic profile comparable to that of human IgG with a similarisotype. Immunoglobulins of the IgG class are among the most abundantproteins in human blood, and their circulation half-lives can reach aslong as 21 days. To extend the circulating half-life of IL-15 or anIL-15 fusion protein and/or to increase its biological activity, fusionprotein complexes containing the IL-15 domain non-covalently bound toIL-15Rα covalently linked to the Fc portion of the human heavy chain IgGprotein are described herein.

The term “Fc” refers to the fragment crystallizable region which is theconstant region of an antibody that interacts with cell surfacereceptors called Fc receptors and some proteins of the complementsystem. Such an “Fc” is in dimeric form. The original immunoglobulinsource of the native Fc is preferably of human origin and may be any ofthe immunoglobulins, although IgG1 and IgG2 are preferred. Native Fc'sare made up of monomeric polypeptides that may be linked into dimeric ormultimeric forms by covalent (i.e., disulfide bonds) and non-covalentassociation. The number of intermolecular disulfide bonds betweenmonomeric subunits of native Fc molecules ranges from 1 to 4 dependingon class (e.g., IgG, IgA, IgE) or subclass (e.g., IgG1, IgG2, IgG3,IgA1, IgGA2). One example of a native Fc is a disulfide-bonded dimerresulting from papain digestion of an IgG (see Ellison et al. (1982),Nucleic Acids Res. 10: 4071-9). The term “native Fc” as used herein isgeneric to the monomeric, dimeric, and multimeric forms. Fc domainscontaining binding sites for Protein A, Protein G, various Fc receptorsand complement proteins. In some embodiments, Fc domain of the complexis capable of interacting with Fc receptors to mediateantibody-dependent cell-mediated cytotoxicity (ADCC) and/or antibodydependent cellular phagocytosis (ADCP). In other applications, thecomplex comprises an Fc domain (e.g., IgG4 Fc) that is incapable ofeffectively mediating ADCC or ADCP.

In some embodiments, the term “Fc variant” refers to a molecule orsequence that is modified from a native Fc, but still comprises abinding site for the salvage receptor, FcRn. International applicationsWO 97/34631 and WO 96/32478 describe exemplary Fc variants, as well asinteraction with the salvage receptor, and are hereby incorporated byreference. Thus, the term “Fc variant” comprises a molecule or sequencethat is humanized from a non-human native Fc. Furthermore, a native Fccomprises sites that may be removed because they provide structuralfeatures or biological activity that are not required for the fusionmolecules of the present invention. Thus, in certain embodiments, theterm “Fc variant” comprises a molecule or sequence that alters one ormore native Fc sites or residues that affect or are involved in (1)disulfide bond formation, (2) incompatibility with a selected host cell(3) N-terminal heterogeneity upon expression in a selected host cell,(4) glycosylation, (5) interaction with complement, (6) binding to an Fcreceptor other than a salvage receptor, (7) antibody-dependent cellularcytotoxicity (ADCC) or (8) antibody-dependent cellular phagocytosis(ADCP). Such alterations can increase or decrease any one or more ofthese Fc properties. Fc variants are described in further detailhereinafter.

The term “Fc domain” encompasses native Fc and Fc variant molecules andsequences as defined above. As with Fc variants and native Fc's, theterm “Fc domain” includes molecules in monomeric or multimeric form,whether digested from whole antibody or produced by recombinant geneexpression or by other means.

Linkers

In some cases, the fusion complexes of the invention also include aflexible linker sequence interposed between the IL-15 or IL-15Rα domainsand the IL-12 and/or IL-18 binding domain. The linker sequence shouldallow effective positioning of the polypeptide with respect to the IL-15or IL-15Rα domains to allow functional activity of both domains.

In certain cases, the soluble fusion protein complex has a linkerwherein the first polypeptide is covalently linked to IL-15 (orfunctional fragment thereof) by polypeptide linker sequence. In otheraspects, the soluble fusion protein complex as described herein has alinker wherein the second polypeptide is covalently linked to IL-15Rαpolypeptide (or functional fragment thereof) by polypeptide linkersequence.

The linker sequence is preferably encoded by a nucleotide sequenceresulting in a peptide that can effectively position the binding grooveof a TCR molecule for recognition of a presenting antigen or the bindingdomain of an antibody molecule for recognition of an antigen. As usedherein, the phrase “effective positioning of the biologically activepolypeptide with respect to the IL-15 or IL-15Rα domains”, or othersimilar phrase, is intended to mean the biologically active polypeptidelinked to the IL-15 or IL-15Rα domains is positioned so that the IL-15or IL-15Rα domains are capable of interacting with each other to form aprotein complex. For example, the IL-15 or IL-15Rα domains areeffectively positioned to allow interactions with immune cells toinitiate or inhibit an immune reaction, or to inhibit or stimulate celldevelopment.

The fusion complexes of the invention preferably also include a flexiblelinker sequence interposed between the IL-15 or IL-15Rα domains and theimmunoglobulin Fc domain. The linker sequence should allow effectivepositioning of the Fc domain, biologically active polypeptide and IL-15or IL-15Rα domains to allow functional activity of each domain. Forexample, the Fc domains are effectively positioned to allow properfusion protein complex formation and/or interactions with Fc receptorson immune cells or proteins of the complement system to stimulateFc-mediated effects including opsonization, cell lysis, degranulation ofmast cells, basophils, and eosinophils, and other Fc receptor-dependentprocesses; activation of the complement pathway; and enhanced in vivohalf-life of the fusion protein complex.

Linker sequences can also be used to link two or more polypeptides ofthe biologically active polypeptide to generate a single-chain moleculewith the desired functional activity.

Preferably, the linker sequence comprises from about 7 to 20 aminoacids, more preferably from about 10 to 20 amino acids. The linkersequence is preferably flexible so as not hold the biologically activepolypeptide or effector molecule in a single undesired conformation. Thelinker sequence can be used, e.g., to space the recognition site fromthe fused molecule. Specifically, the peptide linker sequence can bepositioned between the biologically active polypeptide and the effectormolecule, e.g., to chemically cross-link same and to provide molecularflexibility. The linker preferably predominantly comprises amino acidswith small side chains, such as glycine, alanine and serine, to providefor flexibility. Preferably, about 80 or 90 percent or greater of thelinker sequence comprises glycine, alanine or serine residues,particularly glycine and serine residues.

Different linker sequences could be used including any of a number offlexible linker designs that have been used successfully to joinantibody variable regions together (see, Whitlow, M. et al., (1991)Methods: A Companion to Methods in Enzymology, 2:97-105).

Adoptive Cell Therapy

Adoptive cell therapy (ACT) (including allogeneic and autologoushematopoietic stem cell transplantation (HSCT) and recombinant cell(i.e., CAR T) therapies) is the treatment of choice for many malignantdisorders (for reviews of HSCT and adoptive cell therapy approaches,see, Rager & Porter, Ther Adv Hematol (2011) 2(6) 409-428; Roddie &Peggs, Expert Opin. Biol. Ther. (2011) 11(4):473-487; Wang et al. Int.J. Cancer: (2015)136, 1751-1768; and Chang, Y. J. and X. J. Huang, BloodRev, 2013. 27(1): 55-62). Such adoptive cell therapies include, but arenot limited to, allogeneic and autologous hematopoietic stem celltransplantation, donor leukocyte (or lymphocyte) infusion (DLI),adoptive transfer of tumor infiltrating lymphocytes, or adoptivetransfer of T cells or NK cells (including recombinant cells, i.e., CART, CAR NK). Beyond the necessity for donor-derived cells to reconstitutehematopoiesis after radiation and chemotherapy, immunologicreconstitution from transferred cells is important for the eliminationof residual tumor cells. The efficacy of ACT as a curative option formalignancies is influenced by a number of factors including the origin,composition and phenotype (lymphocyte subset, activation status) of thedonor cells, the underlying disease, the pre-transplant conditioningregimen and post-transplant immune support (i.e., IL-2 therapy) and thegraft-versus-tumor (GVT) effect mediated by donor cells within thegraft. Additionally, these factors must be balanced againsttransplant-related mortality, typically arising from the conditioningregimen and/or excessive immune activity of donor cells within the host(i.e., graft-versus-host disease, cytokine release syndrome, etc.).

Approaches utilizing adoptive NK cell therapy have become of significantinterest. In patients receiving autologous HSCT, blood NK cell numbersrecover very early after the transplant and the levels of NK cellscorrelate with a positive outcome (Rueff et al., 2014, Biol. BloodMarrow Transplant. 20, 896-899). Although therapeutic strategies withautologous NK cell transfer have had limited success due to a number offactors, adoptive transfer of ex vivo activated allogeneic (orhaplo-identical) NK cells has emerged as a promising immunotherapeuticstrategy for cancer (Guillerey et al. 2016. Nature Immunol. 17:1025-1036). The activity of these cells is less likely to be suppressedby self-MHC molecules compared to autologous NK cells. A number ofstudies have shown that adoptive therapy with haploidentical NK cells toexploit alloreactivity against tumor cells is safe and can mediatesignificant clinical activity in AML patients. Taking these findingsfurther, recent studies have focused on optimizing ex vivoactivation/expansion methods for NK cells or NK precursors (i.e., stemcells) and pre-transplant conditioning and post-transplant immunesupport strategies; use of NK cell lines or recombinant tumor-targetingNK cells; evaluation of combination therapies with other agents such astherapeutic Ab, immunomodulatory agents (lenalidomide), and anti-KIR andcheckpoint Abs. In each case, these strategies could be complemented bythe fusion protein complex of the invention, which has the capacity toaugment NK cell proliferation and activation. As indicated herein, exvivo incubation of NK cells with the fusion protein complex of theinvention result in induction of CIML NK cell exhibiting elevatedactivation markers, increased cytotoxicity against tumor cells andenhanced production of IFN-γ. Additionally, the fusion protein complexof the invention is capable of activating human NK cell lines. Moreover,methods are provided for augmenting immune responses and treatingneoplasia and infection disease by direct administration of the fusionprotein complex of the invention or administration of immune cellsactivated by the fusion protein complex of the invention.

Natural Killer Cells: One of the major types of circulating mononuclearcells is that of the natural killer, or NK, cell (M. Manoussaka et al.,Journal of Immunology 158:112-119, 1997). Originally defined based ontheir ability to kill certain tumors and virus-infected cells, NK cellsare now known as one of the components of the early, innate immunesystem. In addition to their cytotoxic capabilities, NK cells serve asregulators of the immune response by releasing a variety of cytokines.In addition, the generation of complex immune responses is facilitatedby the direct interaction of NK cells with other cells via varioussurface molecules expressed on the NK cells.

NK cells are derived from bone marrow precursors (O. Haller et al.,Journal of Experimental Medicine 145:1411-1420, 1977). NK cells appearto be closely related to T cells, and the two cell types share many cellsurface markers (M. Manoussaka et al., 1997). As noted above, these cellsurface markers play a significant role in NK cell activity. Forexample, murine NK cells express specific antigens on their surfaces,such as asialo GM1, NK1, and NK2 antigens (D. See et al., Scand. J.Immunol. 46:217-224, 1997), and the administration of antibodies againstthese antigens results in depletion of NK cells in vivo (Id.).

Similarly to cytotoxic T lymphocytes (CTL), NK cells exert a cytotoxiceffect by lysing a variety of cell types (Srivastava, S., Lundqvist, A.& Childs, R. W. Natural killer cell immunotherapy for cancer: a newhope. Cytotherapy 10, 775-783; 2008). These include normal stem cells,infected cells, and transformed cells. The lysis of cells occurs throughthe action of cytoplasmic granules containing proteases, nucleases, andperforin. Cells that lack MHC class I are also susceptible to NKcell-mediated lysis (H. Reyburn et al., Immunol. Rev. 155:119-125,1997). In addition, NK cells exert cytotoxicity in a non-MHC restrictedfashion (E. Ciccione et al., J. Exp. Med. 172:47, 1990; A. Moretta etal., J. Exp. Med. 172:1589, 1990; and E. Ciccione et al., J. Exp. Med.175:709). NK cells can also lyse cells by antibody-dependent cellularcytotoxicity.

As noted above, NK cells mediate some of their functions through thesecretion of cytokines, such as interferon γ (IFN-γ),granulocyte-macrophage colony-stimulating factors (GM-CSFs), tumornecrosis factor α (TNF-α), macrophage colony-stimulating factor (M-CSF),interleukin-3 (IL-3), and IL-8. NK cell cytotoxic activity is regulatedthrough a balance of activating and inhibitory receptors that enablesfine-tuned control of cytotoxic activity, preventing cytotoxicityagainst healthy cells, while maintaining effective cytotoxic capacityagainst tumor cells. Indeed, multiple studies have demonstrated thesafety of adoptive NK cell transfer and clinical anti-cancer effects,highlighting the potential for NK cells as an effective cancerimmunotherapy ((Parkhurst, M. R., et al. Clin Cancer Res 17, 6287-6297(2011); Ruggeri, L. et al. Science 295, 2097-2100, (2002); Miller, J. S.et al. Blood 105, 3051-3057, (2005; Bachanova, V. et al. Blood 123,3855-3863, (2014); Rubnitz, J. E. et al. J Clin Oncol 28, 955-959,(2010)). For example, cytokines including IL-2, IL-12, TNF-α, and IL-1can induce NK cells to produce cytokines. IFN-α and IL-2 are stronginducers of NK cell cytotoxic activity (G. Trinichieri et al., Journalof Experimental Medicine 160:1147-1169, 1984; G. Trinichieri and D.Santoli, Journal of Experimental Medicine 147: 1314-1333, 1977). Thepresence of IL-2 both stimulates and expands NK cells (K. Oshimi,International Journal of Hematology 63:279-290, 1996). IL-12 has beenshown to induce cytokine production from T and NK cells, and augment NKcell-mediated cytotoxicity (M. Kobayashi et al., Journal of ExperimentalMedicine 170:827-846, 1989).

NK cells are involved in both the resistance to and control of cancerspread. Since the advent of the cancer immune surveillance concept, theadoptive transfer of immune cells, particularly T cells and naturalkiller (NK) cells, has emerged as a targeted method of harnessing theimmune system against cancer (Kroemer, G., Senovilla, L., Galluzzi, L.,Andre, F. & Zitvogel, L. Natural and therapy-induced immunosurveillancein breast cancer. Nat Med 21, 1128-1138, (2015)). NK cells have garneredimmense attention as a promising immunotherapeutic agent for treatingcancers. NK cells are critical to the body's first line of defenseagainst cancer due to their natural cytotoxicity against malignant cells(Srivastava, S., et al., Cytloherapy 10, 775-783; 2008).

NK cells have been expanded from multiple sources, including peripheralblood and umbilical cord blood (CB) ((Denman, C. J. et al.Membrane-bound IL-21 promotes sustained ex vivo proliferation of humannatural killer cells. PLoS One 7, e30264, (2012); Knorr, D. A. et al.Clinical-scale derivation of natural killer cells from human pluripotentstem cells for cancer therapy. Stem Cells Transi Med 2, 274-283, (2013);Shah, N. et al. Antigen presenting cell-mediated expansion of humanumbilical cord blood yields log-scale expansion of natural killer cellswith anti-myeloma activity. PLoS One 8, e76781, (2013); Wol, P. S. etal. Human embryonic stem cells differentiate into a homogeneouspopulation of natural killer cells with potent in vivo antitumoractivity. Blood 113, 6094-6101, (2009)). Ex vivo NK cell expansionmethods have been developed using cytokines in combination withartificial antigen-presenting cells (aAPCs) as feeder cells ((Denman, C.J. et al. PLoS One 7, e30264, (2012); Berg, M. et al. Cytotherapy 11,341-355, (2009); Gong, W. et al. Tissue Antigens 76, 467-475, (2010);Zhang, H. et al., J Immunother 34, 187-195, (2011)).

Cytokine-Based Therapies in Senescent Cell- and Aging-RelatedPathologies

Damaged cells undergo either apoptosis or senescence. Senescent cellsprevent their own proliferation and secrete signaling molecules—aphenomenon known as the senescence-associated secretory phenotype (SASP)(Coppe J. P. et al., 2010 Annu Rev Pathol 5:99-118). It is proposed thatthe SASP is to restore tissue function by stimulating less-damagedneighboring cells to engage tissue repair by attracting immune cells(Demaria M., et al., 2014 Dev. Cell 31:722-733). These immune cellseliminate the senescent cells to turn off SASP-mediated signals. Whendamage exceeds repair capacity or immune cells become unresponsive toeffects of the SASP, the aberrant accumulation of senescent cellsoccurs. As a result, senescent cells accumulate in aged and/or damagedorgans and aggravate tissue dysfunction (Ovadya Y. and Krizhanovsky V.et al., 2014 Biogerontology 15:627-642). The elimination of senescentcells has been shown to increase healthy lifespan and reduce theseverity of age-related diseases in mice (Baar M. P. et al., 2017 Cell169:132-147; Baker D J 2016 530:184-189).

Thus, physiological aging is associated with the appearance of senescentcells. Evidence suggests that senescent cells compromise tissuehomeostasis and function, and their accumulation contributes to thedevelopment of age-associated pathologies (Baker D J et al., 2008 NatCell Biol 10:825-836; Baker D J et al., 2016, 530:184-189). In additionto a shorten life span, senescent cells are associated with pathologiesinclude metabolic (obesity, diabetes), neurological (Alzheimer's andParkinson's diseases), muscle, bone, and cartilage related (sarcopenia,osteoarthritis, kyphosis, herniated discs) or tissue dysfunction related(lung emphysema, cardiovascular and renal diseases, and atherosclerosis)diseases. Studies have shown that the innated and adaptive immunesystems are involved in the recognition and elimination of senescentcells (Soto-Gamez A. and Demaria M., 2017 Drug Discovery Today22:786-795; Hazeldine J. and Lord, J. M., 2013 Aging Research Reviews12:1069-1078). For instance, NK cells were demonstrated to eliminatesenescent cells via the granule exocytosis pathway. Therefore,augmenting these responses may increase natural mechanisms of senescencesurveillance and reduce senescent cell-associated pathologies. It hasalso been suggested that the age-related decline in perforin-mediated NKcell cytotoxicity is responsible in part for the increased frequency ofsenescent cells in aged tissue (Rukavina D. et al., Blood 92:2410-2420;Sagiv A. et al. 2012 Oncogene 1-7). In liver fibrosis, the accumulatedof senescent activated stellate cells is increased in mice lacking NKG2Dreceptor, the major activating receptor of NK cells, leading toincreased fibrosis (Sagiv A. et al., 2016 Aging 8:328-344).

The IL-7/IL-21/TxM complexes of the invention have been demonstrated tohave potent activity to enhance the cytotoxicity of both the innate andadaptive immune cells including NK and T cells. As disclosed herein, theIL-7/IL-21/TxM complexes are expected to activate and/or maintain immuneresponses against senescent cells and to have potential applications asanti-aging therapeutic agents. As indicated, the IL-7/IL-21/TxMcomplexes have advantages over individual cytokines in providing morepotent immune stimulation to both NK and T cells. In addition, immunecells stimulated ex vivo by IL-7/IL-21/TxM complexes could be used inadoptive cell transplant for treatment of senescent cell- and/orage-related diseases. Such therapies could be adoptive NK or T celltherapies. Administration of IL-7/IL-21/TxM complexes following adoptivecell transplant could also be conducted to support proliferation,activation, and persistence of the transferred cells.

Pharmaceutical Therapeutics

The invention provides pharmaceutical compositions comprising fusionprotein complexes for use as a therapeutic. In one aspect, fusionprotein complex of the invention is administered systemically, forexample, formulated in a pharmaceutically-acceptable buffer such asphysiological saline. Preferable routes of administration include, forexample, instillation into the bladder, subcutaneous, intravenous,intraperitoneal, intramuscular, intratumoral or intradermal injectionsthat provide continuous, sustained or effective levels of thecomposition in the patient. Treatment of human patients or other animalsis carried out using a therapeutically effective amount of a therapeuticidentified herein in a physiologically-acceptable carrier. Suitablecarriers and their formulation are described, for example, inRemington's Pharmaceutical Sciences by E. W. Martin. The amount of thetherapeutic agent to be administered varies depending upon the manner ofadministration, the age and body weight of the patient, and with theclinical symptoms of the neoplasia. Generally, amounts will be in therange of those used for other agents used in the treatment of otherdiseases associated with neoplasia, autoimmune or infectious diseases,although in certain instances lower amounts will be needed because ofthe increased specificity of the compound. A compound is administered ata dosage that enhances an immune response of a subject, or that reducesthe proliferation, survival, or invasiveness of a neoplastic, infected,autoimmune or senescent cell as determined by a method known to oneskilled in the art.

Formulation of Pharmaceutical Compositions

The administration of the fusion protein complex of the invention forthe treatment of a neoplasia, infectious, senescent cell- or age-relatedor autoimmune disease is by any suitable means that results in aconcentration of the therapeutic that, combined with other components,is effective in ameliorating, reducing, or stabilizing said neoplasia,infectious, senescent cell- or age-related or autoimmune disease. Thefusion protein complex of the invention may be contained in anyappropriate amount in any suitable carrier substance, and is generallypresent in an amount of 1-95% by weight of the total weight of thecomposition. The composition may be provided in a dosage form that issuitable for parenteral (e.g., subcutaneous, intravenous, intramuscular,intravesicular, intratumoral or intraperitoneal) administration route.For example, the pharmaceutical compositions are formulated according toconventional pharmaceutical practice (see, e.g., Remington: The Scienceand Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, LippincottWilliams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology,eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York).

Human dosage amounts are initially determined by extrapolating from theamount of compound used in mice or non-human primates, as a skilledartisan recognizes it is routine in the art to modify the dosage forhumans compared to animal models. For example, the dosage may vary frombetween about 1 μg compound/kg body weight to about 5000 mg compound/kgbody weight; or from about 5 mg/kg body weight to about 4,000 mg/kg bodyweight or from about 10 mg/kg body weight to about 3,000 mg/kg bodyweight; or from about 50 mg/kg body weight to about 2000 mg/kg bodyweight; or from about 100 mg/kg body weight to about 1000 mg/kg bodyweight; or from about 150 mg/kg body weight to about 500 mg/kg bodyweight. For example, the dose is about 1, 5, 10, 25, 50, 75, 100, 150,200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850,900, 950, 1,000, 1,050, 1,100, 1,150, 1,200, 1,250, 1,300, 1,350, 1,400,1,450, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, 3,000, 3,500,4,000, 4,500, or 5,000 mg/kg body weight. Alternatively, doses are inthe range of about 5 mg compound/Kg body weight to about 20 mgcompound/kg body weight. In another example, the doses are about 8, 10,12, 14, 16 or 18 mg/kg body weight. Preferably, the fusion proteincomplex is administered at 0.5 mg/kg-about 10 mg/kg (e.g., 0.5, 1, 3, 5,10 mg/kg). Of course, this dosage amount may be adjusted upward ordownward, as is routinely done in such treatment protocols, depending onthe results of the initial clinical trials and the needs of a particularpatient.

Pharmaceutical compositions are formulated with appropriate excipientsinto a pharmaceutical composition that, upon administration, releasesthe therapeutic in a controlled manner. Examples include single ormultiple unit tablet or capsule compositions, oil solutions,suspensions, emulsions, microcapsules, microspheres, molecularcomplexes, nanoparticles, patches, and liposomes. Preferably, the fusionprotein complex is formulated in an excipient suitable for parenteraladministration.

Parenteral Compositions

The pharmaceutical composition comprising a fusion protein complex ofthe invention are administered parenterally by injection, infusion orimplantation (subcutaneous, intravenous, intramuscular, intratumoral,intravesicular, intraperitoneal) in dosage forms, formulations, or viasuitable delivery devices or implants containing conventional, non-toxicpharmaceutically acceptable carriers and adjuvants. The formulation andpreparation of such compositions are well known to those skilled in theart of pharmaceutical formulation. Formulations can be found inRemington: The Science and Practice of Pharmacy, supra.

Compositions comprising a fusion protein complex of the invention forparenteral use are provided in unit dosage forms (e.g., in single-doseampoules). Alternatively, the composition is provided in vialscontaining several doses and in which a suitable preservative may beadded (see below). The composition is in the form of a solution, asuspension, an emulsion, an infusion device, or a delivery device forimplantation, or it is presented as a dry powder to be reconstitutedwith water or another suitable vehicle before use. Apart from the activeagent that reduces or ameliorates a neoplasia, infectious, senescentcell- or age-related or autoimmune disease, the composition includessuitable parenterally acceptable carriers and/or excipients. The activetherapeutic agent(s) may be incorporated into microspheres,microcapsules, nanoparticles, liposomes for controlled release.Furthermore, the composition may include suspending, solubilizing,stabilizing, pH-adjusting agents, tonicity adjusting agents, and/ordispersing, agents.

As indicated above, the pharmaceutical compositions comprising a fusionprotein complex of the invention may be in a form suitable for sterileinjection. To prepare such a composition, the suitable activetherapeutic(s) are dissolved or suspended in a parenterally acceptableliquid vehicle. Among acceptable vehicles and solvents that may beemployed are water, water adjusted to a suitable pH by addition of anappropriate amount of hydrochloric acid, sodium hydroxide or a suitablebuffer, 1,3-butanediol, Ringer's solution, and isotonic sodium chloridesolution and dextrose solution. The aqueous formulation may also containone or more preservatives (e.g., methyl, ethyl or n-propylp-hydroxybenzoate). In cases where one of the compounds is onlysparingly or slightly soluble in water, a dissolution enhancing orsolubilizing agent can be added, or the solvent may include 10-60% w/wof propylene glycol.

The present invention provides methods of treating neoplasia, infectiousdiseases, senescent cell- or age-related diseases or symptoms thereofwhich comprise administering a therapeutically effective amount of apharmaceutical composition comprising a compound of the formulae hereinto a subject (e.g., a mammal such as a human). Thus, one embodiment is amethod of treating a subject suffering from or susceptible to aneoplasia, infectious disease, senescent cell- or age-related diseasesor symptom thereof. The method includes the step of administering to themammal a therapeutic amount of an amount of a compound herein sufficientto treat the disease or disorder or symptom thereof, under conditionssuch that the disease or disorder is treated.

The methods herein include administering to the subject (including asubject identified as in need of such treatment) an effective amount ofa compound described herein, or a composition described herein toproduce such effect. Identifying a subject in need of such treatment canbe in the judgment of a subject or a health care professional and can besubjective (e.g. opinion) or objective (e.g. measurable by a test ordiagnostic method).

The therapeutic methods of the invention (which include prophylactictreatment) in general comprise administration of a therapeuticallyeffective amount of the compounds herein, such as a compound of theformulae herein to a subject (e.g., animal, human) in need thereof,including a mammal, particularly a human. Such treatment will besuitably administered to subjects, particularly humans, suffering from,having, susceptible to, or at risk for a neoplasia, infectious disease,senescent cell- or age-related diseases, disorder, or symptom thereof.Determination of those subjects “at risk” can be made by any objectiveor subjective determination by a diagnostic test or opinion of a subjector health care provider (e.g., genetic test, enzyme or protein marker,Marker (as defined herein), family history, and the like). The fusionprotein complexes of the invention may be used in the treatment of anyother disorders in which an increase in an immune response is desired.

The invention also provides a method of monitoring treatment progress.The method includes the step of determining a level of diagnostic marker(Marker) (e.g., any target delineated herein modulated by a compoundherein, a protein or indicator thereof, etc.) or diagnostic measurement(e.g., screen, assay) in a subject suffering from or susceptible to adisorder or symptoms thereof associated with neoplasia in which thesubject has been administered a therapeutic amount of a compound hereinsufficient to treat the disease or symptoms thereof. The level of Markerdetermined in the method can be compared to known levels of Marker ineither healthy normal controls or in other afflicted patients toestablish the subject's disease status. In some cases, a second level ofMarker in the subject is determined at a time point later than thedetermination of the first level, and the two levels are compared tomonitor the course of disease or the efficacy of the therapy. In certainaspects, a pre-treatment level of Marker in the subject is determinedprior to beginning treatment according to this invention; thispre-treatment level of Marker can then be compared to the level ofMarker in the subject after the treatment commences, to determine theefficacy of the treatment.

Combination Therapies

Optionally, the fusion protein complex of the invention is administeredin combination with any other standard therapy; such methods are knownto the skilled artisan and described in Remington's PharmaceuticalSciences by E. W. Martin. If desired, fusion protein complexes of theinvention is administered in combination with any conventionalanti-neoplastic therapy, including but not limited to, immunotherapy,therapeutic antibodies, targeted therapy, surgery, radiation therapy, orchemotherapy.

Kits or Pharmaceutical Systems

Pharmaceutical compositions comprising the fusion protein complex of theinvention may be assembled into kits or pharmaceutical systems for usein ameliorating a neoplasia, infectious disease or senescent cell- orage-related diseases. Kits or pharmaceutical systems according to thisaspect of the invention comprise a carrier means, such as a box, carton,tube, having in close confinement therein one or more container means,such as vials, tubes, ampoules, bottles and the like. The kits orpharmaceutical systems of the invention may also comprise associatedinstructions for using the fusion protein complex of the invention.

Recombinant Protein Expression

In general, preparation of the fusion protein complexes of the invention(e.g., components of a TxM complex) can be accomplished by proceduresdisclosed herein and by recognized recombinant DNA techniques.

In general, recombinant polypeptides are produced by transformation of asuitable host cell with all or part of a polypeptide-encoding nucleicacid molecule or fragment thereof in a suitable expression vehicle.Those skilled in the field of molecular biology will understand that anyof a wide variety of expression systems may be used to provide therecombinant protein. The precise host cell used is not critical to theinvention. A recombinant polypeptide may be produced in virtually anyeukaryotic host (e.g., Saccharomyces cerevisiae, insect cells, e.g.,Sf21 cells, or mammalian cells, e.g., NIH 3T3, HeLa, or preferably COScells). Such cells are available from a wide range of sources (e.g., theAmerican Type Culture Collection, Rockland, Md.; also, see, e.g.,Ausubel et al., Current Protocol in Molecular Biology, New York: JohnWiley and Sons, 1997). The method of transfection and the choice ofexpression vehicle will depend on the host system selected.Transformation methods are described, e.g., in Ausubel et al. (supra);expression vehicles may be chosen from those provided, e.g., in CloningVectors: A Laboratory Manual (P. H. Pouwels et al., 1985, Supp. 1987).

A variety of expression systems exist for the production of recombinantpolypeptides. Expression vectors useful for producing such polypeptidesinclude, without limitation, chromosomal, episomal, and virus-derivedvectors, e.g., vectors derived from bacterial plasmids, frombacteriophage, from transposons, from yeast episomes, from insertionelements, from yeast chromosomal elements, from viruses such asbaculoviruses, papova viruses, such as SV40, vaccinia viruses,adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses,and vectors derived from combinations thereof.

Once the recombinant polypeptide is expressed, it is isolated, e.g.,using affinity chromatography. In one example, an antibody (e.g.,produced as described herein) raised against the polypeptide may beattached to a column and used to isolate the recombinant polypeptide.Lysis and fractionation of polypeptide-harboring cells prior to affinitychromatography may be performed by standard methods (see, e.g., Ausubelet al., supra). Once isolated, the recombinant protein can, if desired,be further purified, e.g., by high performance liquid chromatography(see, e.g., Fisher, Laboratory Techniques in Biochemistry and MolecularBiology, eds., Work and Burdon, Elsevier, 1980).

As used herein, biologically active polypeptides or effector moleculesof the invention may include factors such as cytokines, chemokines,growth factors, protein toxins, immunoglobulin domains or otherbioactive proteins such as enzymes. Also, biologically activepolypeptides may include conjugates to other compounds such asnon-protein toxins, cytotoxic agents, chemotherapeutic agents,detectable labels, radioactive materials and such.

Cytokines of the invention are defined by any factor produced by cellsthat affect other cells and are responsible for any of a number ofmultiple effects of cellular immunity. Examples of cytokines include butare not limited to the IL-2 family, interferon (IFN), IL-10, IL-12,IL-18, IL-1, IL-17, TGF and TNF cytokine families, and to IL-1 throughIL-35, IFN-α, IFN-β, IFN-γ, TGF-β, TNF-α, and TNF-β.

In an aspect of the invention, the first protein comprises a firstbiologically active polypeptide covalently linked to interleukin-15(IL-15) domain or a functional fragment thereof. IL-15 is a cytokinethat affects T-cell activation and proliferation. IL-15 activity inaffecting immune cell activation and proliferation is similar in somerespects to IL-2, although fundamental differences have been wellcharacterized (Waldmann, T A, 2006, Nature Rev. Immunol. 6:595-601).

In another aspect of the invention, the first protein comprises aninterleukin-15 (IL-15) domain that is an IL-15 variant (also referred toherein as IL-15 mutant). The IL-15 variant preferably comprises adifferent amino acid sequence that the native (or wild type) IL-15protein. The IL-15 variant preferably binds the IL-15Rα polypeptide andfunctions as an IL-15 agonist or antagonist. Preferably, IL-15 variantswith agonist activity have super agonist activity. The IL-15 variant canfunction as an IL-15 agonist or antagonist independent of itsassociation with IL-15Rα. IL-15 agonists are exemplified by comparableor increased biological activity compared to wild type IL-15. IL-15antagonists are exemplified by decreased biological activity compared towild type IL-15 or by the ability to inhibit IL-15-mediated responses.In some examples, the IL-15 variant binds with increased or decreasedactivity to the IL-15RβγC receptors. In some cases, the sequence of theIL-15 variant has at least one amino acid change, e.g. substitution ordeletion, compared to the native IL-15 sequence, such changes resultingin IL-15 agonist or antagonist activity. Preferably, the amino acidsubstitutions/deletions are in the domains of IL-15 that interact withIL-15Rβ and/or γC. More preferably, the amino acidsubstitutions/deletions do not affect binding to the IL-15Rα polypeptideor the ability to produce the IL-15 variant. Suitable amino acidsubstitutions/deletions to generate IL-15 variants can be identifiedbased on putative or known IL-15 structures, comparisons of IL-15 withhomologous molecules such as IL-2 with known structure, through rationalor random mutagenesis and functional assays, as provided herein, orother empirical methods. Additionally, suitable amino acid substitutionscan be conservative or non-conservative changes and insertions ofadditional amino acids. Preferably, IL-15 variants of the inventioncontain one or more than one amino acid substitutions/deletions atposition 6, 8, 10, 61, 65, 72, 92, 101, 104, 105, 108, 109, 111, or 112of the mature human IL-15 sequence; particularly, D8N (“D8” refers tothe amino acid and residue position in the native mature human IL-15sequence and “N” refers to the substituted amino acid residue at thatposition in the IL-15 variant), I6S, D8A, D61A, N65A, N72R, V104P orQ108A substitutions result in IL-15 variants with antagonist activityand N72D substitutions result in IL-15 variants with agonist activity.

Chemokines, like cytokines, are defined as any chemical factor ormolecule which when exposed to other cells are responsible for manyeffects of cellular immunity. Suitable chemokines may include but arenot limited to the CXC, CC, C, and CX₃C chemokine families and to CCL-1through CCL-28, CXC-1 through CXC-17, XCL-1, XCL-2, CX3CL1, MIP-1b,IL-8, MCP-1, and Rantes.

Growth factors include any molecules which when exposed to a particularcell induce proliferation and/or differentiation of the affected cell.Growth factors include proteins and chemical molecules, some of whichinclude: GM-CSF, G-CSF, human growth factor and stem cell growth factor.Additional growth factors may also be suitable for uses describedherein.

Toxins or cytotoxic agents include any substance that has a lethaleffect or an inhibitory effect on growth when exposed to cells. Morespecifically, the effector molecule can be a cell toxin of, e.g., plantor bacterial origin such as, e.g., diphtheria toxin (DT), shiga toxin,abrin, cholera toxin, ricin, saporin, pseudomonas exotoxin (PE),pokeweed antiviral protein, or gelonin. Biologically active fragments ofsuch toxins are well known in the art and include, e.g., DT A chain andricin A chain. Additionally, the toxin can be an agent active at thecell surface such as, e.g., phospholipase enzymes (e.g., phospholipaseC).

Further, the effector molecule can be a chemotherapeutic drug such as,e.g., vindesine, vincristine, vinblastin, methotrexate, adriamycin,bleomycin, or cisplatin.

Additionally, the effector molecule can be a detectably-labeled moleculesuitable for diagnostic or imaging studies. Such labels include biotinor streptavidin/avidin, a detectable nanoparticles or crystal, an enzymeor catalytically active fragment thereof, a fluorescent label such asgreen fluorescent protein, FITC, phycoerythrin, cychome, texas red orquantum dots; a radionuclide e.g., iodine-131, yttrium-90, rhenium-188or bismuth-212; a phosphorescent or chemiluminescent molecules or alabel detectable by PET, ultrasound or MRI such as Gd—or paramagneticmetal ion-based contrast agents. See e.g., Moskaug, et al. J. Biol.Chem. 264, 15709 (1989); Pastan, I. et al. Cell 47, 641, 1986; Pastan etal., Recombinant Toxins as Novel Therapeutic Agents, Ann. Rev. Biochem.61, 331, (1992); “Chimeric Toxins” Olsnes and Phil, Pharmac. Ther., 25,355 (1982); published PCT application no. WO 94/29350; published PCTapplication no. WO 94/04689; published PCT application no. WO2005046449and U.S. Pat. No. 5,620,939 for disclosure relating to making and usingproteins comprising effectors or tags.

The IL-15 and IL-15Rα polypeptides of the invention suitably correspondin amino acid sequence to naturally occurring IL-15 and IL-15Rαmolecules, e.g. IL-15 and IL-15Rα molecules of a human, mouse or otherrodent, or other mammal. Sequences of these polypeptides and encodingnucleic acids are known in the literature, including human interleukin15 (IL15) mRNA—GenBank: U14407.1 (incorporated herein by reference), Musmusculus interleukin 15 (IL15) mRNA—GenBank: U14332.1 (incorporatedherein by reference), human interleukin-15 receptor alpha chainprecursor (IL15RA) mRNA—GenBank: U31628.1 (incorporated herein byreference), Mus musculus interleukin 15 receptor, alpha chain—GenBank:BC095982.1 (incorporated herein by reference).

In some settings, it can be useful to make the protein fusion orconjugate complexes of the present invention polyvalent, e.g., toincrease the valency of the sc-antibody. In particular, interactionsbetween the IL-15 and IL-15Rα domains of the fusion protein complexprovide a means of generating polyvalent complexes. In addition, thepolyvalent fusion protein can be made by covalently or non-covalentlylinking together between one and four proteins (the same or different)by using e.g., standard biotin-streptavidin labeling techniques, or byconjugation to suitable solid supports such as latex beads. Chemicallycross-linked proteins (for example cross-linked to dendrimers) are alsosuitable polyvalent species. For example, the protein can be modified byincluding sequences encoding tag sequences that can be modified such asthe biotinylation BirA tag or amino acid residues with chemicallyreactive side chains such as Cys or His. Such amino acid tags orchemically reactive amino acids may be positioned in a variety ofpositions in the fusion protein, preferably distal to the active site ofthe biologically active polypeptide or effector molecule. For example,the C-terminus of a soluble fusion protein can be covalently linked to atag or other fused protein which includes such a reactive amino acid(s).Suitable side chains can be included to chemically link two or morefusion proteins to a suitable dendrimer or other nanoparticle to give amultivalent molecule. Dendrimers are synthetic chemical polymers thatcan have any one of a number of different functional groups of theirsurface (D. Tomalia, Aldrichimica Acta, 26:91:101 (1993)). Exemplarydendrimers for use in accordance with the present invention include e.g.E9 starburst polyamine dendrimer and E9 combust polyamine dendrimer,which can link cysteine residues. Exemplary nanoparticles includeliposomes, core-shell particles or PLGA-based particles.

In another aspect, one or both of the polypeptides of the fusion proteincomplex comprises an immunoglobulin domain. Alternatively, the proteinbinding domain-IL-15 fusion protein can be further linked to animmunoglobulin domain. The preferred immunoglobulin domains compriseregions that allow interaction with other immunoglobulin domains to formmultichain proteins as provided above. For example, the immunoglobulinheavy chain regions, such as the IgG1 C_(H)2-C_(H)3, are capable ofstably interacting to create the Fc region. Preferred immunoglobulindomains including Fc domains also comprise regions with effectorfunctions, including Fc receptor or complement protein binding activity,and/or with glycosylation sites. In some aspects, the immunoglobulindomains of the fusion protein complex contain mutations that reduce oraugment Fc receptor or complement binding activity or glycosylation ordimerization, thereby affecting the biological activity of the resultingprotein. For example, immunoglobulin domains containing mutations thatreduce binding to Fc receptors could be used to generate fusion proteincomplex of the invention with lower binding activity to Fcreceptor-bearing cells, which may be advantageous for reagents designedto recognize or detect specific antigens.

Nucleic Acids and Vectors

The invention further provides nucleic acid sequences and particularlyDNA sequences that encode the present fusion proteins (e.g., componentsof TxM). Preferably, the DNA sequence is carried by a vector suited forextrachromosomal replication such as a phage, virus, plasmid, phagemid,cosmid, YAC, or episome. In particular, a DNA vector that encodes adesired fusion protein can be used to facilitate preparative methodsdescribed herein and to obtain significant quantities of the fusionprotein. The DNA sequence can be inserted into an appropriate expressionvector, i.e., a vector that contains the necessary elements for thetranscription and translation of the inserted protein-coding sequence. Avariety of host-vector systems may be utilized to express theprotein-coding sequence. These include mammalian cell systems infectedwith virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systemsinfected with virus (e.g., baculovirus); microorganisms such as yeastcontaining yeast vectors, or bacteria transformed with bacteriophageDNA, plasmid DNA or cosmid DNA. Depending on the host-vector systemutilized, any one of a number of suitable transcription and translationelements may be used. See, Sambrook et al., supra and Ausubel et al.supra.

Included in the invention are methods for making a soluble fusionprotein complex, the method comprising introducing into a host cell aDNA vector as described herein encoding the first and second proteins,culturing the host cell in media under conditions sufficient to expressthe fusion proteins in the cell or the media and allow associationbetween IL-15 domain of a first protein and the soluble IL-15Rα domainof a second protein to form the soluble fusion protein complex,purifying the soluble fusion protein complex from the host cells ormedia.

In general, a preferred DNA vector according to the invention comprisesa nucleotide sequence linked by phosphodiester bonds comprising, in a 5′to 3′ direction a first cloning site for introduction of a firstnucleotide sequence encoding a biologically active polypeptide,operatively linked to a sequence encoding an effector molecule.

The fusion protein components encoded by the DNA vector can be providedin a cassette format. By the term “cassette” is meant that eachcomponent can be readily substituted for another component by standardrecombinant methods. In particular, a DNA vector configured in acassette format is particularly desirable when the encoded fusioncomplex is to be used against pathogens that may have or have capacityto develop serotypes.

To make the vector coding for a fusion protein complex, the sequencecoding for the biologically active polypeptide is linked to a sequencecoding for the effector peptide by use of suitable ligases. DNA codingfor the presenting peptide can be obtained by isolating DNA from naturalsources such as from a suitable cell line or by known synthetic methods,e.g. the phosphate triester method. See, e.g., OligonucleotideSynthesis, IRL Press (M. J. Gait, ed., 1984). Synthetic oligonucleotidesalso may be prepared using commercially available automatedoligonucleotide synthesizers. Once isolated, the gene coding for thebiologically active polypeptide can be amplified by the polymerase chainreaction (PCR) or other means known in the art. Suitable PCR primers toamplify the biologically active polypeptide gene may add restrictionsites to the PCR product. The PCR product preferably includes splicesites for the effector peptide and leader sequences necessary for properexpression and secretion of the biologically active polypeptide-effectorfusion complex. The PCR product also preferably includes a sequencecoding for the linker sequence, or a restriction enzyme site forligation of such a sequence.

The fusion proteins described herein are preferably produced by standardrecombinant DNA techniques. For example, once a DNA molecule encodingthe biologically active polypeptide is isolated, sequence can be ligatedto another DNA molecule encoding the effector polypeptide. Thenucleotide sequence coding for a biologically active polypeptide may bedirectly joined to a DNA sequence coding for the effector peptide or,more typically, a DNA sequence coding for the linker sequence asdiscussed herein may be interposed between the sequence coding for thebiologically active polypeptide and the sequence coding for the effectorpeptide and joined using suitable ligases. The resultant hybrid DNAmolecule can be expressed in a suitable host cell to produce the fusionprotein complex. The DNA molecules are ligated to each other in a 5′ to3′ orientation such that, after ligation, the translational frame of theencoded polypeptides is not altered (i.e., the DNA molecules are ligatedto each other in-frame). The resulting DNA molecules encode an in-framefusion protein.

Other nucleotide sequences also can be included in the gene construct.For example, a promoter sequence, which controls expression of thesequence coding for the biologically active polypeptide fused to theeffector peptide, or a leader sequence, which directs the fusion proteinto the cell surface or the culture medium, can be included in theconstruct or present in the expression vector into which the constructis inserted. An immunoglobulin or CMV promoter is particularlypreferred.

In obtaining variant biologically active polypeptide, IL-15, IL-15Rα orFc domain coding sequences, those of ordinary skill in the art willrecognize that the polypeptides may be modified by certain amino acidsubstitutions, additions, deletions, and post-translationalmodifications, without loss or reduction of biological activity. Inparticular, it is well-known that conservative amino acid substitutions,that is, substitution of one amino acid for another amino acid ofsimilar size, charge, polarity and conformation, are unlikely tosignificantly alter protein function. The 20 standard amino acids thatare the constituents of proteins can be broadly categorized into fourgroups of conservative amino acids as follows: the nonpolar(hydrophobic) group includes alanine, isoleucine, leucine, methionine,phenylalanine, proline, tryptophan and valine; the polar (uncharged,neutral) group includes asparagine, cysteine, glutamine, glycine,serine, threonine and tyrosine; the positively charged (basic) groupcontains arginine, histidine and lysine; and the negatively charged(acidic) group contains aspartic acid and glutamic acid. Substitution ina protein of one amino acid for another within the same group isunlikely to have an adverse effect on the biological activity of theprotein. In other instance, modifications to amino acid positions can bemade to reduce or enhance the biological activity of the protein. Suchchanges can be introduced randomly or via site-specific mutations basedon known or presumed structural or functional properties of targetedresidue(s). Following expression of the variant protein, the changes inthe biological activity due to the modification can be readily assessedusing binding or functional assays.

Homology between nucleotide sequences can be determined by DNAhybridization analysis, wherein the stability of the double-stranded DNAhybrid is dependent on the extent of base pairing that occurs.Conditions of high temperature and/or low salt content reduce thestability of the hybrid, and can be varied to prevent annealing ofsequences having less than a selected degree of homology. For instance,for sequences with about 55% G-C content, hybridization and washconditions of 40-50 C, 6×SSC (sodium chloride/sodium citrate buffer) and0.1% SDS (sodium dodecyl sulfate) indicate about 60-70% homology,hybridization and wash conditions of 50-65 C, 1×SSC and 0.1% SDSindicate about 82-97% homology, and hybridization and wash conditions of52 C, 0.1×SSC and 0.1% SDS indicate about 99-100% homology. A wide rangeof computer programs for comparing nucleotide and amino acid sequences(and measuring the degree of homology) are also available, and a listproviding sources of both commercially available and free software isfound in Ausubel et al. (1999). Readily available sequence comparisonand multiple sequence alignment algorithms are, respectively, the BasicLocal Alignment Search Tool (BLAST) (Altschul et al., 1997) and ClustalWprograms. BLAST is available on the world wide web at ncbi.nlm.nih.govand a version of ClustalW is available at 2.ebi.ac.uk.

The components of the fusion protein can be organized in nearly anyorder provided each is capable of performing its intended function. Forexample, in one embodiment, the biologically active polypeptide issituated at the C or N terminal end of the effector molecule.

Preferred effector molecules of the invention will have sizes conduciveto the function for which those domains are intended. The effectormolecules of the invention can be made and fused to the biologicallyactive polypeptide by a variety of methods including well-known chemicalcross-linking methods. See, e.g., Means, G. E. and Feeney, R. E. (1974)in Chemical Modification of Proteins, Holden-Day. See also, S. S. Wong(1991) in Chemistry of Protein Conjugation and Cross-Linking, CRC Press.However it is generally preferred to use recombinant manipulations tomake the in-frame fusion protein.

As noted, a fusion molecule or a conjugate molecule in accord with theinvention can be organized in several ways. In an exemplaryconfiguration, the C-terminus of the biologically active polypeptide isoperatively linked to the N-terminus of the effector molecule. Thatlinkage can be achieved by recombinant methods if desired. However, inanother configuration, the N-terminus of the biologically activepolypeptide is linked to the C-terminus of the effector molecule.

Alternatively, or in addition, one or more additional effector moleculescan be inserted into the biologically active polypeptide or conjugatecomplexes as needed.

Vectors and Expression

A number of strategies can be employed to express the components offusion protein complex of the invention (e.g., TxM). For example, aconstruct encoding one or more components of fusion protein complex ofthe invention can be incorporated into a suitable vector usingrestriction enzymes to make cuts in the vector for insertion of theconstruct followed by ligation. The vector containing the gene constructis then introduced into a suitable host for expression of the fusionprotein. See, generally, Sambrook et al., supra. Selection of suitablevectors can be made empirically based on factors relating to the cloningprotocol. For example, the vector should be compatible with, and havethe proper replicon for the host that is being employed. The vector mustbe able to accommodate the DNA sequence coding for the fusion proteincomplex that is to be expressed. Suitable host cells include eukaryoticand prokaryotic cells, preferably those cells that can be easilytransformed and exhibit rapid growth in culture medium. Specificallypreferred hosts cells include prokaryotes such as E. coli, Bacillussubtillus, etc. and eukaryotes such as animal cells and yeast strains,e.g., S. cerevisiae. Mammalian cells are generally preferred,particularly J558, NSO, SP2-O or CHO. Other suitable hosts include,e.g., insect cells such as Sf9. Conventional culturing conditions areemployed. See, Sambrook, supra. Stable transformed or transfected celllines can then be selected. Cells expressing a fusion protein complex ofthe invention can be determined by known procedures. For example,expression of a fusion protein complex linked to an immunoglobulin canbe determined by an ELISA specific for the linked immunoglobulin and/orby immunoblotting. Other methods for detecting expression of fusionproteins comprising biologically active polypeptides linked to IL-15 orIL-15Rα domains are disclosed in the Examples.

As mentioned generally above, a host cell can be used for preparativepurposes to propagate nucleic acid encoding a desired fusion protein.Thus, a host cell can include a prokaryotic or eukaryotic cell in whichproduction of the fusion protein is specifically intended. Thus hostcells specifically include yeast, fly, worm, plant, frog, mammaliancells and organs that are capable of propagating nucleic acid encodingthe fusion. Non-limiting examples of mammalian cell lines which can beused include CHO dhfr-cells (Urlaub and Chasm, Proc. Natl. Acad. Sci.USA, 77:4216 (1980)), 293 cells (Graham et al., J Gen. Virol., 36:59(1977)) or myeloma cells like SP2 or NSO (Galfre and Milstein, Meth.Enzymol., 73(B):3 (1981)).

Host cells capable of propagating nucleic acid encoding a desired fusionprotein complexes encompass non-mammalian eukaryotic cells as well,including insect (e.g., Sp. frugiperda), yeast (e.g., S. cerevisiae, S.pombe, P. pastoris., K. lactis, H. polymorpha; as generally reviewed byFleer, R., Current Opinion in Biotechnology, 3(5):486496 (1992)), fungaland plant cells. Also contemplated are certain prokaryotes such as E.coli and Bacillus.

Nucleic acid encoding a desired fusion protein can be introduced into ahost cell by standard techniques for transfecting cells. The term“transfecting” or “transfection” is intended to encompass allconventional techniques for introducing nucleic acid into host cells,including calcium phosphate co-precipitation, DEAE-dextran-mediatedtransfection, lipofection, electroporation, microinjection, viraltransduction and/or integration. Suitable methods for transfecting hostcells can be found in Sambrook et al. supra, and other laboratorytextbooks.

Various promoters (transcriptional initiation regulatory region) may beused according to the invention. The selection of the appropriatepromoter is dependent upon the proposed expression host. Promoters fromheterologous sources may be used as long as they are functional in thechosen host.

Promoter selection is also dependent upon the desired efficiency andlevel of peptide or protein production. Inducible promoters such as tacare often employed to dramatically increase the level of proteinexpression in E. coli. Overexpression of proteins may be harmful to thehost cells. Consequently, host cell growth may be limited. The use ofinducible promoter systems allows the host cells to be cultivated toacceptable densities prior to induction of gene expression, therebyfacilitating higher product yields.

Various signal sequences may be used according to the invention. Asignal sequence which is homologous to the biologically activepolypeptide coding sequence may be used. Alternatively, a signalsequence which has been selected or designed for efficient secretion andprocessing in the expression host may also be used. For example,suitable signal sequence/host cell pairs include the B. subtilis sacBsignal sequence for secretion in B. subtilis, and the Saccharomycescerevisiae α-mating factor or P. pastoris acid phosphatase phoI signalsequences for P. pastoris secretion. The signal sequence may be joineddirectly through the sequence encoding the signal peptidase cleavagesite to the protein coding sequence, or through a short nucleotidebridge consisting of usually fewer than ten codons, where the bridgeensures correct reading frame of the downstream TCR sequence.

Elements for enhancing transcription and translation have beenidentified for eukaryotic protein expression systems. For example,positioning the cauliflower mosaic virus (CaMV) promoter 1,000 bp oneither side of a heterologous promoter may elevate transcriptionallevels by 10- to 400-fold in plant cells. The expression constructshould also include the appropriate translational initiation sequences.Modification of the expression construct to include a Kozak consensussequence for proper translational initiation may increase the level oftranslation by 10 fold.

A selective marker is often employed, which may be part of theexpression construct or separate from it (e.g., carried by theexpression vector), so that the marker may integrate at a site differentfrom the gene of interest. Examples include markers that conferresistance to antibiotics (e.g., bla confers resistance to ampicillinfor E. coli host cells, nptlI confers kanamycin resistance to a widevariety of prokaryotic and eukaryotic cells) or that permit the host togrow on minimal medium (e.g., HIS4 enables P. pastoris or His⁻ S.cerevisiae to grow in the absence of histidine). The selectable markerhas its own transcriptional and translational initiation and terminationregulatory regions to allow for independent expression of the marker. Ifantibiotic resistance is employed as a marker, the concentration of theantibiotic for selection will vary depending upon the antibiotic,generally ranging from 10 to 600 μg of the antibiotic/mL of medium.

The expression construct is assembled by employing known recombinant DNAtechniques (Sambrook et al., 1989; Ausubel et al., 1999). Restrictionenzyme digestion and ligation are the basic steps employed to join twofragments of DNA. The ends of the DNA fragment may require modificationprior to ligation, and this may be accomplished by filling in overhangs,deleting terminal portions of the fragment(s) with nucleases (e.g.,ExoII), site directed mutagenesis, or by adding new base pairs by PCR.Polylinkers and adaptors may be employed to facilitate joining ofselected fragments. The expression construct is typically assembled instages employing rounds of restriction, ligation, and transformation ofE. coli. Numerous cloning vectors suitable for construction of theexpression construct are known in the art (λZAP and pBLUESCRIPT SK-1,Stratagene, La Jolla, Calif., pET, Novagen Inc., Madison, Wis., cited inAusubel et al., 1999) and the particular choice is not critical to theinvention. The selection of cloning vector will be influenced by thegene transfer system selected for introduction of the expressionconstruct into the host cell. At the end of each stage, the resultingconstruct may be analyzed by restriction, DNA sequence, hybridizationand PCR analyses.

The expression construct may be transformed into the host as the cloningvector construct, either linear or circular, or may be removed from thecloning vector and used as is or introduced onto a delivery vector. Thedelivery vector facilitates the introduction and maintenance of theexpression construct in the selected host cell type. The expressionconstruct is introduced into the host cells by any of a number of knowngene transfer systems (e.g., natural competence, chemically mediatedtransformation, protoplast transformation, electroporation, biolistictransformation, transfection, or conjugation) (Ausubel et al., 1999;Sambrook et al., 1989). The gene transfer system selected depends uponthe host cells and vector systems used.

For instance, the expression construct can be introduced into S.cerevisiae cells by protoplast transformation or electroporation.Electroporation of S. cerevisiae is readily accomplished, and yieldstransformation efficiencies comparable to spheroplast transformation.

The present invention further provides a production process forisolating a fusion protein of interest. In the process, a host cell(e.g., a yeast, fungus, insect, bacterial or animal cell), into whichhas been introduced a nucleic acid encoding the protein of the interestoperatively linked to a regulatory sequence, is grown at productionscale in a culture medium to stimulate transcription of the nucleotidessequence encoding the fusion protein of interest. Subsequently, thefusion protein of interest is isolated from harvested host cells or fromthe culture medium. Standard protein purification techniques can be usedto isolate the protein of interest from the medium or from the harvestedcells. In particular, the purification techniques can be used to expressand purify a desired fusion protein on a large-scale (i.e. in at leastmilligram quantities) from a variety of implementations including rollerbottles, spinner flasks, tissue culture plates, bioreactor, or afermenter.

An expressed protein fusion complex can be isolated and purified byknown methods. Typically the culture medium is centrifuged or filteredand then the supernatant is purified by affinity or immunoaffinitychromatography, e.g. Protein-A or Protein-G affinity chromatography oran immunoaffinity protocol comprising use of monoclonal antibodies thatbind the expressed fusion complex. The fusion proteins of the presentinvention can be separated and purified by appropriate combination ofknown techniques. These methods include, for example, methods utilizingsolubility such as salt precipitation and solvent precipitation, methodsutilizing the difference in molecular weight such as dialysis,ultra-filtration, gel-filtration, and SDS-polyacrylamide gelelectrophoresis, methods utilizing a difference in electrical chargesuch as ion-exchange column chromatography, methods utilizing specificaffinity such as affinity chromatography, methods utilizing a differencein hydrophobicity such as reverse-phase high performance liquidchromatography and methods utilizing a difference in isoelectric point,such as isoelectric focusing electrophoresis, metal affinity columnssuch as Ni-NTA. See generally Sambrook et al. and Ausubel et al. suprafor disclosure relating to these methods.

It is preferred that the fusion proteins of the present invention besubstantially pure. That is, the fusion proteins have been isolated fromcell substituents that naturally accompany it so that the fusionproteins are present preferably in at least 80% or 90% to 95%homogeneity (w/w). Fusion proteins having at least 98 to 99% homogeneity(w/w) are most preferred for many pharmaceutical, clinical and researchapplications. Once substantially purified the fusion protein should besubstantially free of contaminants for therapeutic applications. Oncepurified partially or to substantial purity, the soluble fusion proteinscan be used therapeutically, or in performing in vitro or in vivo assaysas disclosed herein. Substantial purity can be determined by a varietyof standard techniques such as chromatography and gel electrophoresis.

The present fusion protein complexes are suitable for in vitro or invivo use with a variety of cells that are cancerous or are infected orthat may become infected by one or more diseases.

Human interleukin-15 (huIL-15) is trans-presented to immune effectorcells by the human IL-15 receptor α chain (huIL-15Rα) expressed onantigen presenting cells. IL-15Rα binds huIL-15 with high affinity (38pM) primarily through the extracellular sushi domain (huIL-15RαSu). Asdescribed herein, the huIL-15 and huIL-15RαSu domains can be used as ascaffold to construct multi-domain fusion complexes.

IgG domains, particularly the Fc fragment, have been used successfullyas dimeric scaffolds for a number of therapeutic molecules includingapproved biologic drugs. For example, etanercept is a dimer of solublehuman p75 tumor necrosis factor-α (TNF-α) receptor (sTNFR) linked to theFc domain of human IgG1. This dimerization allows etanercept to be up to1,000 times more potent at inhibiting TNF-α activity than the monomericsTNFR and provides the fusion with a five-fold longer serum half-lifethan the monomeric form. As a result, etanercept is effective atneutralization of the pro-inflammatory activity of TNF-α in vivo andimproving patient outcome for a number of different autoimmuneindications.

In addition to its dimerization activity, the Fc fragment also providescytotoxic effector functions through the complement activation andinteraction with Fcγ receptors displayed on natural killer (NK) cells,neutrophils, phagocytes and dendritic cells. In the context ofanti-cancer therapeutic antibodies and other antibody domain-Fc fusionproteins, these activities likely play an important role in efficacyobserved in animal tumor models and in cancer patients. However thesecytotoxic effector responses may not be sufficient in a number oftherapeutic applications. Thus, there has been considerable interest inimproving and expanding on the effector activity of the Fc domain anddeveloping other means of recruiting cytolytic immune responses,including T cell activity, to the disease site via targeted therapeuticmolecules. IgG domains have been used as a scaffold to form bispecificantibodies to improve the quality and quantity of products generated bythe traditional hybridoma fusion technology. Although these methodsbypass the shortcomings of other scaffolds, it has been difficult toproduce bispecific antibodies in mammalian cells at levels sufficient tosupport clinical development and use.

In an effort to develop human-derived immunostimulatory multimericscaffold, human IL-15 (huIL-15) and IL-15 receptor domains were used.huIL-15 is a member of the small four α-helix bundle family of cytokinesthat associates with the huIL-15 receptor α-chain (huIL-15Rα) with ahigh binding affinity (equilibrium dissociation constant (KD)˜10⁻¹¹ M).The resulting complex is then trans-presented to the human IL-2/15receptor β/common γ chain (huIL-15RβγC) complexes displayed on thesurface of T cells and NK cells. This cytokine/receptor interactionresults in expansion and activation of effector T cells and NK cells,which play an important role in eradicating virally infected andmalignant cells. Normally, huIL-15 and huIL-15Rα are co-produced indendritic cells to form complexes intracellularly that are subsequentlysecreted and displayed as heterodimeric molecules on cell surfaces.Thus, the characteristics of huIL-15 and huIL-15Rα interactions suggestthat these inter chain binding domains could serve as a human-derivedimmunostimulatory scaffold to make soluble dimeric molecules capable oftarget-specific binding.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are well within the purview of the skilled artisan.Such techniques are explained fully in the literature, such as,“Molecular Cloning: A Laboratory Manual”, second edition (Sambrook,1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture”(Freshney, 1987); “Methods in Enzymology” “Handbook of ExperimentalImmunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells”(Miller and Calos, 1987); “Current Protocols in Molecular Biology”(Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994);“Current Protocols in Immunology” (Coligan, 1991). These techniques areapplicable to the production of the polynucleotides and polypeptides ofthe invention, and, as such, may be considered in making and practicingthe invention. Particularly useful techniques for particular embodimentswill be discussed in the sections that follow.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the assay, screening, and therapeutic methods of theinvention, and are not intended to limit the scope of what the inventorsregard as their invention.

EXAMPLES Example 1: Generation and Characterization of Fusion ProteinComplexes Comprising IL-15, IL-7 and IL-21 Domains

An important therapeutic approach for treating cancer or infectiousdisease relies on augmenting immune cell activity against the diseasedcells. This strategy includes stimulating immune cells ex vivo followedby adoptive transfer and/or directly increasing immune cell levels oractivity in vivo in the patient. Immune cells involved in theseapproaches may be those of the innate (i.e., NK cells) or adaptive(i.e., T cells) immune system.

One approach for augmenting immune activity is to provideimmunostimulatory cytokines to the immune cells. Such cytokines areknown in the art and can be used alone or in combination with othercytokines or agents. As described in detail below, fusion proteincomplexes comprising an IL-15N72D:IL-15RαSu/Fc scaffold fused to IL-7and IL-21 binding domains were generated (FIGS. 1A, 1B). These fusionprotein complexes have advantages in binding to NK and T cells andsignaling cell responses via each of the cytokine receptors. The Fcregion of Ig molecules forms a dimer to provide a solublemulti-polypeptide complex, can bind Protein A for the purpose ofpurification and can interact with Fcγ receptors on NK cells andmacrophages, thus providing advantages to the fusion protein complexthat are not present in the combination of individual cytokines.Additionally, interactions between the IL-15N72D and IL-15RαSu domainsprovide a means to link the IL-15N72D, IL-7 and IL-21 (and possiblyother protein domains or agents) into a single immunostimulatory fusionprotein complex.

Specifically, constructs were made linking IL-7 and IL-21 domains to theIL-15N72D and IL-15RαSu/Fc chains. In some cases, either IL-7 or IL-21polypeptide is linked to the N-terminus of the IL-15N72D and/orIL-15RαSu/Fc chains. In other cases, the IL-7 or IL-21 polypeptide islinked to the N-terminus of IL-15N72D and/or IL-15RαSu/Fc chains.Specific fusion protein complexes comprising an IL-15N72D:IL-15RαSu/Fcscaffold fused to IL-7 and IL-21 binding domains are described below.

1) A fusion protein complex was generated comprising IL-21/IL-15RαSu/Fcand IL-7/IL-15N72D fusion proteins. The human IL-7 and human IL-21sequences were obtained from the UniProt website and DNA for thesesequences was synthesized by Genewiz. Specifically, constructs were madedirectly linking the synthesized IL-21 sequence to the N-terminal codingregion of IL-15RαSu/Fc via overlapping PCR. The nucleic acid and proteinsequences of a construct comprising IL-21 linked to the N-terminus ofIL-15RαSu/Fc are shown below.

The nucleic acid sequence of the IL-21/IL-15RαSu/Fc construct (includingsignal peptide sequence) is as follows (SEQ ID NO: 1):

(Signal peptide) atgaagtgggtgaccttcatcagcctgctgttcctgttctccagcgccta ctcc(Human IL-21) cagggccaggacaggcacatgatccggatgaggcagctcatcgacatcgtcgaccagctgaagaactacgtgaacgacctggtgcccgagtttctgcctgcccccgaggacgtggagaccaactgcgagtggtccgccttctcctgctttcagaaggcccagctgaagtccgccaacaccggcaacaacgagcggatcatcaacgtgagcatcaagaagctgaagcggaagcctccctccacaaacgccggcaggaggcagaagcacaggctgacctgccccagctgtgactcctacgagaagaagccccccaaggagttcctggagaggttcaagtccctgctgcagaagatgatccatcagcacctgtcctccaggacccacggctccgaggactcc(Human IL-15R a sushi domain)atcacgtgtcctcctcctatgtccgtggaacacgcagacatctgggtcaagagctacagcttgtactccagggagcggtacatttgtaactctggtttcaagcgtaaagccggcacgtccagcctgacggagtgcgtgttgaacaaggccacgaatgtcgcccactggacaacccccagtctcaaatgcattaga(Human IgG1 CH2-CH3 (Fc) domain)gagccgaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctcctggtaaa

The amino acid sequence of the IL-21/IL-15RαSu/Fc construct (includingsignal peptide sequence) is as follows (SEQ ID NO: 2):

(Signal peptide) MKWVTFISLLFLFSSAYS (Human IL-21)QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS (Human IL-15R a sushi domain)ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKA TNVAHWTTPSLKCIR(Human IgG1 CH2-CH3 (Fc) domain)EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

In some cases, the leader peptide is cleaved from the intact polypeptideto generate the mature form that may be soluble or secreted.

Constructs were also made linking the synthesized IL-7 sequence to theN-terminus coding region of IL-15N72D via overlapping PCR. The nucleicacid and protein sequences of a construct comprising IL-7 linked to theN-terminus of IL-15N72D are shown below.

The nucleic acid sequence of the IL-7/IL-15N72D construct (includingleader sequence) is as follows (SEQ ID NO: 3):

(Signal peptide) atgaagtgggtgaccttcatcagcctgctgttcctgttctccagcgccta ctcc(Human IL-7) gattgcgacatcgagggcaaggacggcaagcagtacgagagcgtgctgatggtgtccatcgaccagctgctggacagcatgaaggagatcggctccaactgcctcaacaacgagttcaacttcttcaagcggcacatctgcgacgccaacaaggagggcatgttcctgttcagggccgccaggaaactgcggcagttcctgaagatgaactccaccggcgacttcgacctgcacctgctgaaggtgtccgagggcaccaccatcctgctgaactgcaccggacaggtgaagggccggaaacctgctgctctgggagaggcccaacccaccaagagcctggaggagaacaagtccctgaaggagcagaagaagctgaacgacctgtgcttcctgaagaggctgctgcaggagatcaagacctgctggaacaagatcctgatgggcaccaag gagcat(Human IL-15N72D) aactgggttaacgtaataagtgatttgaaaaaaattgaagatcttattcaatctatgcatattgatgctactttatatacggaaagtgatgttcaccccagttgcaaagtaacagcaatgaagtgctttctcttggagttacaagttatttcacttgagtccggagatgcaagtattcatgatacagtagaaaatctgatcatcctagcaaacgacagtttgtcttctaatgggaatgtaacagaatctggatgcaaagaatgtgaggaactggaggaaaaaaatattaaagaatttttgcagagttttgtacatattgtccaaatgttcatcaacacttct

The amino acid sequence of the mature IL-7/IL-15N72D fusion protein(including leader sequence) is as follows (SEQ ID NO: 4):

(Signal peptide) MKWVTFISLLFLFSSAYS (Human IL-7)DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTK EH (Human IL-15N72D)NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANDSLSSNGNVTESGCKECEELEEKNIKEFL QSFVHIVQMFINTS

In some cases, the leader peptide is cleaved from the intact polypeptideto generate the mature form that may be soluble or secreted.

The IL-21/IL-15RαSu/Fc and IL-7/IL-15N72D constructs were cloned intoexpression vectors as described previously (U.S. Pat. No. 8,507,222, atExample 1, incorporated herein by reference), and the expression vectorstransfected into CHO cells. Co-expression of the two constructs in CHOcells allowed for formation and secretion of a solubleIL-7/IL-15N72D:IL-21/IL-15RαSu/Fc fusion protein complex (referred to ashIL7/IL21/TxM). The hIL7/IL21/TxM protein was purified from CHO cellculture supernatant by Protein A affinity chromatography and sizeexclusion chromatography resulting in soluble (non-aggregated) fusionprotein complexes consisting of IL-21/IL-15RαSu/Fc dimers andIL-7/IL-15N72D fusion proteins (FIG. 2).

Reduced SDS-PAGE and Western blot analyses of the Protein A-purifiedIL-7/IL-15N72D:IL-21/IL-15RαSu/Fc fusion protein complexes are shown inFIG. 3. For the Western blot analysis, purified proteins were separatedby reduced SDS-PAGE on 4-12% Bis-Tris gel. The resolved protein bandswere transferred from the gel to membrane using the Invitrogen iBlot2system. The membrane was probed with 1) goat anti-hIgG Fc Ab (primary)and rabbit anti-goat IgG-AP (secondary) for detection of protein bandscontaining the human Fc domain and 2) biotinylated mouse anti-hIL15 Ab(primary) and streptavidin-HRP (secondary) for detection of proteinbands containing the human IL-15 domain. Binding of the probe reagentswas detected following incubation with appropriate substrates (NBT/BCIPor TMB) using the Millipore SNAP i.d. 2.0 protein detection system.Bands in the SDS-PA gels and Western blots corresponding to the solubleIL-21/IL-15RαSu/Fc and IL-7/IL-15N72D proteins migrate at about 54 kDaand −38-45 kDa, respectively, were observed (FIGS. 3A and B).

The calculated molecular masses of the IL-21/IL-15RαSu/Fc andIL-7/IL-15N72D proteins are about 49 kDa and 30 kDa, respectively. Toassess the differences between the calculated and observed molecularmasses, deglycosylation studies were conducted on the purified proteincomplexes using the Protein Deglycosylation Mix II (New EnglandBioLabs). Briefly, 100 μg of protein complex in 1× Deglycosylation MixBuffer 2 was denatured at 75° C. for 10 minutes. After cooling down toroom temperature, 5 μl of Protein Deglycosylation Mix II was added tothe protein mixture. Following incubation at room temperature for 30minutes, the proteins were deglycosylated overnight at 37° C. After thedeglycosylation reaction, the samples are ready to be analyzed onreduced Coomassie-stained SDS-PAGE. ALT-803, hIL7/IL21/TxM protein thatwas not deglycosylated, and the Protein Deglycosylation Mix (containingthe deglycosylase enzyme) were also analyzed as controls. The resultsindicate that bands corresponding to the deglycosylatedIL-21/IL-15RαSu/Fc and IL-7/IL-15N72D proteins migrate at about 51 kDaand 30 kDa, respectively, consistent with the calculated molecularmasses (FIG. 4). These findings confirm that the mammalian cell producedproteins of the hIL7/IL21/TxM complex are glycosylated. ELISA-basedmethods confirmed the formation of the hIL7/IL21/TxM fusion proteincomplex. In FIG. 5, the IL-7/IL-15N72D:IL-21/IL-15RαSu/Fc fusion proteincomplexes in the culture supernatant from transfected CHO cells weredetected using a huIgG1/IL15-specific ELISA with a capture antibody,anti-human IL-15 antibody (MAB647, R&D Systems) and a detectionantibody, horseradish peroxidase conjugated anti-human IgG antibody.This is compared to a similar cytokine TxM fusion protein complex(hIL18/IL12/TxM) with a known concentration. The signal from thehIL7/IL21/TxM fusion protein complex can be compared to that of thehIL18/IL12/TxM control to estimate the fusion protein concentration.

Similar ELISAs were conducted on purified hIL7/IL21/TxM using captureand probe antibodies to human IgG (GAH) and IL-15 and combinations ofantibodies to IL7 and IL15, IL21 and IL15 and IL7 and IL21 (FIGS.6A-6E). In these assays, hIL18/IL12/TxM and ALT-803 were used ascontrols. The results from these assays demonstrate that solubleIL-7/IL-15N72D, IL-21/IL-15RαSu/Fc proteins can be produced in CHO cellsand the hIL7/IL21/TxM fusion protein complexes can form and be secretedinto the culture media. The secreted protein can be purified and thefusion protein complex remains intact.

Example 2: In Vitro Characterization of the Activities of hIL7/IL21/TxMFusion Protein Complexes

To assess the IL-15 immunostimulatory activity of the hIL7/IL21/TxMfusion protein complexes, proliferation of IL-15-dependent 32D13 cells,a mouse hematopoietic cell line, was assessed. Increasing levels ofhIL7/IL21/TxM were added to 32D13 cells (10⁴ cells/well) in 200 μL IMDM:10% FBS media and cells were incubated for 3 days at 37° C. PrestoBluecell viability reagent (20 μL/well) then was added. After 4 hours,absorbance was measured at 570 nm (with a 600 nm reference wavelengthfor normalization) to determine cell proliferation based on reduction ofPrestoBlue, a resazurin-based solution, by metabolically active cells.The bioactivity of the IL-15N72D:IL-15RαSu/Fc complex (ALT-803) wasassessed as a positive control. As shown in FIG. 7, hIL7/IL21/TxM wasable to promote cell proliferation of 32D13 cells, thereby demonstratingIL-15 activity. The activity of hIL7/IL21/TxM was reduced compared tothat of ALT-803, possibly due to the linkage of IL-7 to the IL-15N72Ddomain.

In order to demonstrate the individual activity of each cytokine (IL-7,IL-21, and IL-15), flow cytometry-based intracellular phosphoproteinassays were developed by utilizing proteins that are uniquelyphosphorylated in response to receptor signaling by each cytokine (IL-7:Stat5, IL-21: Stat3, and IL-15: Stat5). To test IL-7 activity, mouse 2E8cell lines were cultured in Iscove's modified Dulbecco's medium (IMDM)with 4 mM L-glutamine, 1.5 g/L sodium bicarbonate, 0.05 mM2-mercaptoethanol, 1 ng/ml mouse interleukin-7 and 20% FBS. For thephospho-flow cytometry-based Stat5 assay, cells were washed twice inIMDM and cultured at 0.5×10⁶/ml overnight in complete media without IL-7in a 37° C., 5% CO₂ incubator. The next day, the cells were washed inIMDM and counted.

IL-15 activity was measured in 32D13 cell lines by measuringphospho-Stat5 by flow cytometer. 32D13 cell lines were cultured in IMDMwith 2 ng/ml human IL-2 and 10% FBS. For the phospho-flow cytometrybased Stat5 assay, cell were washed twice in IMDM and cultured at0.5×10⁶/ml overnight in complete media without IL-7 in a 37° C., 5% CO₂incubator. The next day, the cells were washed in IMDM and counted.

IL-21 activity was measured in purified human T cells by measuringphospho-Stat3 (727) by flow cytometer. Fresh human leukocytes wereobtained from the blood bank and CD3⁺ T cells were isolated with theRosetteSep/human CD3⁺ T cell reagent (StemCell Technologies). The purityof CD3⁺ T cells was >95%. Cells were cultured in RPMI 1640 supplementedwith 2 mM L-glutamine, penicillin, streptomycin, and 10% FBS.

Cells (0.25×10⁶/tube) were seeded in FACS tube to test activity of eachcytokine. Cells were stimulated with IL-7 (100 ng), IL-21 (100 ng) andALT-803 (100 ng) alone or in combination at the same concentrations orwith hIL7/IL21/TxM (1000-62.5 ng/mL) for 15 minutes in a 37° C., 5% CO₂incubator. Final volume of media and cytokine were adjusted to 100 μL.After a 15 min incubation, paraformaldehyde (Sigma) was added at a finalconcentration of 1.6% and the cells were incubated in room temperaturein the dark for an additional 10 min. The cells were then washed with 1mL of FACS buffer (PBS, 0.5% BSA, 0.1% NaN₃) by centrifugation at 1500rpm for 5 min at room temperature. The cell pellet was resuspended in100 μl of chilled 100% methanol by gently vortexing. Cells were furtherincubated for 30 min at 4° C. and then washed with 1 mL of FACS buffer.The cell pellets were then resuspended in 50 μl of FACs buffercontaining Phospho-Stat5 Alexa-Fluro-488 antibody (BD Bioscience) totest IL-7 and IL-15 activities or Phospho-Stat3 Alexa-Fluro-488 antibody(BD Bioscience) to test IL-21 activity. After a 30 min incubation atroom temperature in the dark, the cells were washed with 1 mL of FACSbuffer, resuspended in 300 μL of FACS buffer and analyzed by flowcytometry.

Following short term stimulation of mouse 2E8 cells or purified human Tcells (>95% CD3⁺) or 32D13 cells with 1 μg/mL (6.3 nM) hIL7/IL21/TxMresulted in similar responses to that seen with the combination ofrecombinant IL-7 (107 ng/mL; 6.3 nM), IL-21 (112 ng/mL; 6.3 nM) andALT-803 (15.12 ng/mL; 6.3 nM) (FIG. 8A-F). These results demonstratethat each of the cytokine domains of the hIL7/IL21/TxM fusion proteincomplex retains its specific immunostimulatory biological activity.

It is known that the combination of IL-7, IL-21 and IL-15 activity ismore effective at inducing IFN-γ production by naïve T cells than any ofthese cytokines alone. In order to evaluate the combined cytokineactivity of the hIL7/IL21/TxM complex, purified T cells were incubatedwith hIL7/IL21/TxM complex (6.3 nM), combinations of IL-7 (6.3 nM),IL-21 (6.3 nM), and ALT-803 (6.3 nM) or each cytokine alone. After 3days, IFN-γ levels in the culture supernatants were determined by ELISAmethods. As shown in FIG. 9, purified T cells in the presence ofcombined individual cytokines, IL-7+IL-21+ALT-803, induced IFN-γproduction. However, when cultured in the presence of hIL7/IL21/TxMcomplex naïve T cells produced a high level of IFN-γ. These resultsverify that hIL7/IL21/TxM fusion protein complex exhibits the expectedimmunostimulatory activity of the combined IL-7, IL-21 and IL-15cytokines.

Example 3: Proliferation of Purified Naïve T Cells, CD3⁺ T Cells andCD8⁺ T Cells Following Stimulation with hIL7/IL21/TxM Fusion ProteinComplexes

Previous studies have shown that T cell proliferation can be efficientlyinduced in the presence of IL-7, IL-15, and IL-21. To evaluate theability of hIL7/IL21/TxM fusion protein complex to promote proliferationof T cells, human naïve T cells were purified using RosetteSep humannaïve CD8+ T cells kit (STEMCELL Technology) from blood from two healthydonors (1×10⁵ cells/ml). These cells (>85% CD3⁺) were cultured withhIL7/IL21/TxM or the combination of individual recombinant IL-7, IL-21,and ALT-803 for 2-5 days. Proliferation of cytokine-stimulated naïve Tcells was assessed by Presto-Blue and analyzed by flow cytometry. Theresults indicate that hIL7/IL21/TxM fusion protein complex is capable ofinducing proliferation of purified naïve T cells to a similar or greaterextent than the combination of IL-7, IL-21 and IL-15 (FIGS. 10A and Bshow results from 2 different donors, FIG. 10C shows the averagedresults for the 72 hr time-point). Thus, stimulation with thehIL7/IL21/TxM fusion protein complex can induce proliferation ofpurified naïve T cells.

Similarly, enhanced proliferation of T cells was induced ex-vivo bystimulation of purified T cells with IL-7, IL-15, and IL-21. To evaluatethe ability of hIL7/IL21/TxM to promote proliferation of T cells,purified CFSE-labelled human T cells (>90% CD3⁺, purified viaRosetteSep™ Human CD8⁺ T Cell Enrichment Cocktail) (1×10⁵ cells/ml) werestimulated for 7 days with hIL7/IL21/TxM or the combination of separaterecombinant IL-7, IL-21, and ALT-803 and cytokine-induced T cellsproliferation was assessed by flow cytometer. The results indicate thathIL7/IL21/TxM complex was capable of inducing proliferation of purifiedT cells better than the combination of IL-7, IL-21 and IL-15 (FIG. 11).

In further studies, Cell Trace violet-labelled human CD8⁺ T cells (>90%CD8⁺, purified via RosetteSep™ Human CD8⁺ T Cell Enrichment Cocktail)(2.5×10⁵ cells/ml) were stimulated with hIL7/IL21/TxM or the combinationof separate recombinant IL-7, IL-21, and ALT-803. After 7 days, cellswere collected for counting and staining with antibodies to determine Tcell phenotype. Expansion of human CD8+ T cells was greatest following7-day incubation with 10 nM hIL7/IL21/TxM (4.6 fold) compared to amixture of ALT-803 (10 nM), IL-7 (25 ng/mL), and IL-21 (25 ng/mL) (3.9fold) or ALT-803 alone (10 nM) (2.5 fold) (FIG. 12). Cells grown inmedia alone did not show expansion.

Flow cytometry was used to assess the phenotype of CD8 T cellsundergoing cell proliferation as determined by Cell Trace violetdilution (i.e. decreased signal per cell with each division) (FIG. 13).The results confirm the fold expansion data such that the higher levelsof cell proliferation were seen in human CD8⁺ T cells incubated withhIL7/IL21/TxM compared to cells grown in a mixture of ALT-803, IL-7, andIL-21 or ALT-803 alone. No proliferation was observed in cells grown inmedia alone. CD8⁺ T cells expressing CD45RO, CCR7, and CD95 showsignificant proliferation following incubation with hIL7/IL21/TxM.Proliferation was also observed in CD8⁺ T cells with low CCR7 and CD62Lwhereas cells with elevated CD45RA proliferated but to a lesser extentor were converted to the CD45RO phenotype following activation withhIL7/IL21/TxM. The results indicate that proliferation of CD8⁺ centralmemory T cells as well as CD8+ effector memory T cells is induced byhIL7/IL21/TxM and to a greater level than was seen with theALT-803+IL-7+IL-21 combination.

Example 4: In Vitro Expansion of Different Subsets of Human CD8⁺ T Cellsby hIL7/IL21/TxM Fusion Protein Complexes

To assess the in vitro expansion of different subsets of human CD8⁺ Tcells, buffy coat was received from the blood bank. Total CD8 T cellswere isolated with the RosetteSep™ Human CD8 Negative-Selection Kit(STEMCELL Technologies). After enrichment of CD8 T cells, the naïve,central memory, effector memory and memory stem CD8 T cell subsets weresorted by flow cytometry using the following markers. Naïve CD8⁺ T cellswere phenotyped as live CD8⁺, CCR7⁺, CD45RO⁻ and CD95⁻ cells. CD8⁺ Teffector memory cells were phenotyped as live CD8⁺, CCR7⁻, and CD45RO⁺cells. CD8⁺ T central memory cells were phenotyped as live CD8⁺, CCR7⁺,and CD45RO⁺ cells. CD8⁺ T memory stem cells were phenotyped as liveCD8⁺, CCR7⁺, CD45RO⁻, and CD95⁺ cells. Sorted cells were checked forpurity (i.e., samples were considered pure if >95% of the cells had thedesired phenotype).

Sorted different population cells were labelled with CFSE(Carboxyfluorescein succinimidyl ester) (Molecular Probes) as permanufacturer's instruction. The CFSE labelled CD8⁺ T cell subsets werestimulated with either media alone, IL-7+IL-21 (25 ng each), IL7 (25ng)+ALT-803 (144 ng), IL21 (25 ng)+ALT-803 (144 ng), IL7 (25 ng)+IL21(25 ng)+ALT-803 (144 ng) or hIL7/IL21/TxM (1.4 μg) in 200 μL of media ina 96-well flat bottom plate at 37° C., 5% CO₂. After day 7, the cellswere harvested from the wells by washing the wells four times with 2%FBS-PBS and collecting the washes in a tube. The harvested cells werefurther washed once with 2% FBS-PBS by centrifugation at 1500 RPM for 5mins. Cells were resuspended in 100 μL of % FBS-PBS and from that 10 μLwas used for counting cells by hemocytometer following a 1:1 dilutionwith 0.4% Trypan Blue. Cell numbers were determined to assess expansionof the purified CD8⁺ T cell subsets following 7-day incubation with thecytokines or hIL7/IL21/TxM complex. The results indicate that treatmentwith hIL7/IL21/TxM fusion complexes was capable of increasing expansionof naïve, central memory, effector memory and memory stem CD8⁺ T cellsubsets compared incubation to media alone or to combinations of two ofthe tested cytokines. Moreover, hIL7/IL21/TxM complex was more effectivethan the combination of IL-7, IL-21 and ALT-803 at expanding thepopulations of CD8⁺ T cell central memory and effector memory subsets(FIGS. 14A-14D), consistent to the results seen with unsorted human CD8⁺T cells.

In addition to expansion, proliferation of the human CD8⁺ T cell subsetswas assessed based on CSFE dilution by flow cytometry. For thisanalysis, the remaining cells were washed once with 1 ml FACS buffer andcells were stained with anti-CD45RO, CD95, CCR7 and CD8 antibodies (2 μLper sample for 30 minutes in FACS buffer). After 30 minutes at roomtemperature in the dark, the cells were washed with 1 mL FACS buffer andresuspended in 300 μL FACS buffer. Cells were analyzed by flow cytometrygated for different subsets of population. Naïve CD8⁺ T cells werephenotyped as live CD8⁺, CCR7⁺, CD45RO⁻ and CD95⁻ cells. CD8⁺ T effectormemory cells were phenotyped as live CD8⁺, CCR7⁻, and CD45RO⁺ cells.CD8⁺ T central memory cells were phenotyped as live CD8⁺, CCR7⁺, andCD45RO⁺ cells. CD8⁺ T memory stem cells were phenotyped as live CD8⁺,CCR7⁺, CD45RO⁻, and CD95⁺ cells. Each population was evaluated forproliferation based on CSFE dilution. See FIG. 15. This study verifiedthat proliferation of each human CD8 T cell subset was induced byhIL7/IL21/TxM compared to little or no proliferation observed in themedia control cells. Highest levels of cell proliferation were seen inthe CD8⁺ T cell central memory and effector memory subsets followingincubation with hIL7/IL21/TxM compared to all other conditions includingthe ALT-803+IL-7+IL-21 combination.

Example 5: Enhanced Proliferation, Cytotoxicity and Activation ofPurified NK Cells Following Stimulation with hIL7/IL21/TxM FusionProtein Complexes

The ability of hIL7/IL21/TxM to affect proliferation and activation ofhuman NK cells was also evaluated. NK cells were purified humanleukocytes using the StemCell RosetteSep™ Human NK Cell EnrichmentCocktail. Purified NK cells (>90% purity) were seeded at 2×10⁶ cells/mLin 24 well plate in media containing 50 nM hIL7/IL21/TxM, 50 nMhIL18/IL12/TxM (control) or 10 nM ALT-803 (control). After 3 days, cellswere counted and reseeded at 0.5×10⁶ cells/mL with media containingeither 10 nM hIL7/IL21/TxM or 10 nM ALT-803 (control). After anadditional 3 days, cells were again reseeded at 0.5×10⁶ cells/mL withmedia containing either 10 nM hIL7/IL21/TxM or 10 nM ALT-803 (control).On day 10, cells were harvested, assessed for proliferation and cellphenotype and tested for cytotoxicity against K562 target cells.

The results of this study indicated that hIL7/IL21/TxM provided bettercytokine support (day 4-10) for expansion of purified human NK cellsthan ALT-803 regardless of the cytokine used for initial stimulation(day 1-3), such that a majority of the NK cell cultures showed greaterthan a 10-fold expansion by day 10 following growth from day 4-10 inmedia containing hIL7/IL21/TxM (FIG. 16). Based on flow cytometrystaining with NKp46 and CD25 antibodies, the hIL7/IL21/TxM-supported NKcells showed an activated CD25⁺ phenotype comparable to thecytokine-induced memory-like (CIMK) NK cells observed following shortterm incubation with hIL18/IL12/TxM followed by ALT-803 cytokine support(FIG. 17).

The cytotoxic activity of these cells was assessed in 4 hour killingassays with K562 target cells. Briefly, K562 target cells were labeledwith Celltrace violet (CVT) and then co-cultured with expanded human NKeffector cells at a ratio of 2.5:1 (effector:target) for 4 hours. Cellsare harvested washed and resuspended in RPMI containing 2 μg/mLpropidium iodide. Tumor cell lysis was measured by co-staining ofpropidium iodide and CTV. Percent specific killing is calculated bysubtracting the any background death of K562 cells measured from wellsthat were not co-cultured with NK cells. Human NK cells provided aninitial 3-day stimulation with hIL7/IL21/TxM followed by cytokinesupport with either hIL7/IL21/TxM or ALT-803 showed better cytotoxicityagainst K562 cells than those receiving initial hIL18/IL12/TxMstimulation with followed by hIL7/IL21/TxM or ALT-803 cytokine support(FIG. 18). This result was surprising since the activation phenotype ofthese cells appeared to be comparable based on CD25 staining. Togetherthese findings indicate that hIL7/IL21/TxM is highly effective atinducing human NK cell proliferation resulting in an activated phenotypewith elevated cytotoxic activity.

Additional studies were conducted to further characterize the ability ofhIL7/IL21/TxM to induce NK cell cytotoxicity against human tumor cells.Purified human fresh NK cells from 2 different donors were mixed withCellTrace violet-labeled human TF-positive pancreatic cancer SW1990cells for 40 hrs at an effector:target ratio (E:T) of 2:1. ALT-803 orhIL7/IL21/TxM at 50 nM was added to activate the human NK cells andmedia alone served as a control. In some cultures, humanized anti-humantissue factor antibody IgG1 (hOAT) was added at 0.1 nM to induce ADCC.At the end of the incubation period, the percentage of deadviolet-labeled SW1990 cells was determined by staining with propidiumiodide followed by flow cytometry (FIG. 19). Following stimulation withhIL7/IL21/TxM alone, human NK cells exhibited significant cytotoxicityagainst SW1990 tumor cells compared to ALT-803 or control media. Theseresults are consistent with the enhanced cytotoxicity observed withhIL7/IL21/TxM-stimulated NK cell against K562 targets. Addition ofanti-TF antibody resulted in a further increase in NK-mediated ADCC ofthe TF-positive tumor cell, again with NK cells stimulated withhIL7/IL21/TxM showing the highest cytotoxicity. These findings indicatethat hIL7/IL21/TxM provides potent activation of human NK cellsresulting in enhanced natural killing and ADCC against tumor cells.

To assess the potential mechanism of action of hIL7/IL21/TxM, levels ofIFNγ were determine by ELISA using the supernatant of the NK cell/SW1990tumor cell cultures described above. Elevated levels of IFNγ were foundin the NK cell/SW1990 cell cultures containing hIL7/IL21/TxM whereaslittle or no IFNγ was seen in the ALT-803 or control media conditions(FIG. 20). IFNγ release was further induced by addition of the anti-TFantibody to the hIL7/IL21/TxM stimulated cells, but to a much lesserdegree or not at all in the ALT-803 or control media conditions. Theresults indicate that hIL7/IL21/TxM is highly effective at inducing NKcell IFNγ production, which can be further elevated by addition of ADCCantibodies.

The effect of hIL7/IL21/TxM on human NK cell expression of granzyme Bwas also assessed. Purified human NK cells (4×10⁶ cells/well) from 2different donors were incubated in RPMI-10 medium with 50 nMhIL7/IL21/TxM, 50 nM ALT-803 or control media for 16 hours. The NK cellswere then intracellularly stained with FITC-conjugated anti-granzyme Bantibody and levels (MFI-mean fluorescent intensity) of granzyme B wereanalyzed by flow cytometry. Incubation in hIL7/IL21/TxM resulted in a2.8 to 5.3-fold increase in granzyme B levels in NK cells whereas onlymodest increases (1.1 to 1.4-fold) were observed in ALT-803 treated NKcells. See FIG. 21. These findings further exemplify the ability ofhIL7/IL21/TxM to enhance the cytotoxic potential of human NK cells.

Example 6: In Vitro and In Vivo Activity of IL-7/IL-21/TxM Complexes andAdoptive Cell Transfer of IL-7/IL-21/TxM-Stimulated Immune Cells onSenescent Cells and Senescent Cell- and Age-Related Pathologies

As indicated above, accumulation of senescent cells in organs andtissues is associated with age-related diseases. Methods have beendeveloped to evaluate therapeutic strategies for reducing senescentcells and their associated pathologies in vitro and in vivo. To assessthat activity of hIL7/IL21/TxM complexes, senescent cells will begenerated in vitro through methods known in the art. Briefly, humandiploid fibroblasts, IMR-90 and WI38 (ATCC, Manassas, Va., USA), humanforeskin fibroblasts BJ (ATCC, Manassas, Va., USA) and primary humanhepatic myofibroblasts (activated hepatic stellate (HS) cells) will begrown in standard conditions (i.e., DMEM supplemented with 10% FCS, 1%L-Glutamine and 1% penicillin-streptomycin and kept at 37° C. with 7.5%CO₂). DNA damage induced senescent (DIS) cells will be generated bytreating growing cells with Etoposide (100 μM, Sigma) for 48 hours.Cells were considered senescent 7 days after Etoposide removal.Alternatively, oncogene induced senescence will be achieved byretroviral infection of IMR-90 cells with mCherry-H-Rasv12 (or mCherryas a control), and cells were considered senescent 9 days after the endof infection. IMR-90 cells can also be induced to senescence bytreatment twice with 0.1 μM Doxorubicin with a 2-day interval andanalyzed 7 days later. Such cells are representative of chemotherapyinduced senescence. Senescent cells express elevated levels ofbeta-galactosidase which can be assayed as a immunohistochemical (IHC)biomarker to detect these cells in vitro and in tissues (Dimri et al.,Proc. Natl. Acad. Sci. USA. 1995; 92, 9363-9367). Other detectablebiomarkers of senescent cells include p16ink4a, IL-1α (early SASPfactor) and IL-6 (late SASP factor) (Baar et al. Cell. 2017; 169,132-47).

The cytotoxic activity of human NK cell lines (i.e., NK-92) and purifiedhuman NK cells against senescent cells will be assessed in vitro.Briefly, growing or DIS IMR-90 target cells (or growing or DIS WI38, BJor HS cells) will plated in a 12-well plate at 5×105 per well; 10×105NK-92 or purified NK cells will be subsequently added to each well.Following 2 hours of co-incubation, the non-adherent NK cells will bewashed gently and cytotoxicity will be determined based onquantification of remaining adherent target cells using Presto Blue(Life Technologies, CA, USA) (or PI or crystal violet staining)according to the manufacturer's instructions. Based on published results(see for example, Sagiv, et al. Oncogene 2013; 32, 1971-1977), DIS cellsare expected to be more sensitive than growing cells to NK cell-mediatedcytotoxicity at a range of E:T ratios. To assess the effects ofhIL7/IL21/TxM complexes on immune cell activity in this model, human NKcell lines (i.e., NK-92) and purified human NK cells will be incubatedwith various concentrations of hIL7/IL21/TxM complexes or IL-7, IL-21and ALT-803 alone or in combination. The activated NK cells will then beco-incubated at various E:T ratios with growing or DIS target cells asdescribed above and NK-mediated cytotoxicity against the DIS targetswill be determined. It is anticipated that NK cells activated withhIL7/IL21/TxM complexes will exhibit more potent cytotoxicity againstDIS cells that untreated NK cell controls. The selectivity ofhIL7/IL21/TxM-treated NK cells against growing and DIS targets will alsobe evaluated. The results of these studies are anticipated todemonstrate that hIL7/IL21/TxM is an effective “senolytic” agent(defined as an agent that can reduce levels of senescent cells withminimal harm to normal cells). Additionally, hIL7/IL21/TxM-treated NKcells, including adoptively transferred cells, are expected to exhibitsenolytic activity. Similar studies will be carried out with T cells todemonstrate the ability of hIL7/IL21/TxM complexes to stimulate immuneresponses against DIS target cells.

The effects of IL-7/IL-21/TxM complexes on senescent cell- andage-related diseases will be evaluated in animal models. It haspreviously shown the doxorubicin treatment induces senescence in miceresulting in reduced body weight, increased levels of senescent cells inthe liver and decreased liver function as measured by elevated plasmalevels of aspartate aminotransferase (AST) (Baar et al. Cell. 2017; 169,132-47). To evaluate the effects of IL-7/IL-21/TxM complexes in thismodel, 10-40-week-old C57BL6 mice will be treated i.p. with 10 mg/kgdoxorubicin on days 0 and 10 to induce senescence and liver damage.Untreated mice will serve as a control. The mice will then be treatedwith various doses of IL-7/IL-21/TxM complexes (mouse or human versions)or IL-7, IL-21 or ALT-803 alone or in combination. PBS will serve as acontrol treatment. The treatments will be given once or twice weekly byi.v. or s.c. routes starting on day 24. Body weights will be measuredthroughout the treatment period. On day 38, animals will be sacrificedand plasma AST levels will be assessed using an AST Activity Assay Kit(Sigma). Liver sections will also be evaluated for histology and thepresence of senescent cells by IHC using the biomarkers described above.Treatment effects on immune cells (i.e., NK and T cells and subsets)will be assessed in blood, spleen and liver by flow cytometry usingantibodies to detect specific immune cell subsets andactivation/cytotoxicity markers and by methods to detect serum cytokinelevels. Functional activity of treatment-stimulated immune cells (i.e.,from blood or spleen) against senescent cells will be determined usingthe methods described above. Compared to the PBS control group,administration of IL-7/IL-21/TxM complexes is expected to reduce weightloss observed in mice following doxorubicin treatment. Levels of plasmaAST and incidence of senescent cells and lesions in the liver are alsoexpected to be reduced in doxorubicin-treated mice by IL-7/IL-21/TxMtherapy. Corresponding activation of immune responses by IL-7/IL-21/TxMwill provide evidence supporting the immune-mediated mechanism ofaction. Comparison of the treatment effects of IL-7/IL-21/TxM with IL-7,IL-21 or ALT-803 alone or in combination are expected to demonstrate themore potent anti-senescence activity of IL-7/IL-21/TxM therapy.

In addition to evaluation of direct IL-7/IL-21/TxM injection, theanti-senescence activity of adoptively transferredIL-7/IL-21/TxM-stimulated NK or T cells will be evaluated in thedoxorubicin treated C57BL6 mouse model. In this study, mice will betreated with doxorubicin as described above and on day 24, NK or T cellsthat had been treated ex vivo with IL-7/IL-21/TxM complexes (mouse orhuman versions) or IL-7, IL-21 or ALT-803 alone or in combination willbe adoptively transferred into the mice. Further treatment of the micewith IL-7/IL-21/TxM or cytokines may be carried out in some groups toprovide cytokine support to the adoptively transferred cells. Treatmenteffects on immune responses, body weight and doxorubicin-induced liversenescence and damage will be assessed as described above. It isanticipated that adoptive transfer of IL-7/IL-21/TxM-stimulated NK or Tcells will provide significant therapeutic benefit to mice withdoxorubicin-induced senescence.

Further studies of IL-7/IL-21/TxM therapy and adoptive cell transfer ofIL-7/IL-21/TxM-stimulated NK or T cells will be carried out in naturallyaged mice. IL-7/IL-21/TxM complexes or IL-7, IL-21 or ALT-803 alone orin combination will be administered for up to 4 weeks as described aboveto 115-130-week-old (aged) or 26-week-old (young) C57BL6 mice.Alternatively, the activity of adoptively transferredIL-7/IL-21/TxM-stimulated NK or T cells will be evaluated in aged andyoung mice as described above. Changes in body weight, fur density andresponsiveness of the mice to stimuli will be assessed throughout thetreatment period as describe previously (Baar et al. Cell. 2017; 169,132-47). In addition, plasma levels of urea and creatinine will bemeasured with a QuantiChrom Urea Assay Kit (Gentaur) and CreatinineAssay Kit (Sigma), respectively, from samples collected before and aftertreatment as an assessment of age related loss of kidney function.Kidney sections will also be evaluated for the presence of senescentcells as described above. Compared to the PBS control group,administration of IL-7/IL-21/TxM complexes is expected to ameliorateage-related decreases in fur density and inactivity in aged mice. Levelsof plasma urea and creatinine and incidence of senescent cells in thekidneys of aged mice are also expected to be reduced by IL-7/IL-21/TxMtherapy. Similar therapeutic benefits are expected in aged micereceiving adoptively transferred IL-7/IL-21/TxM-stimulated NK or Tcells. Together, the results of these studies are anticipated to showthat treatment with IL-7/IL-21/TxM complex and adoptive cell transfer ofIL-7/IL-21/TxM-stimulated NK or T cells exhibit senolytic activity invivo and reduced senescent cell- and age-related pathologies.

OTHER EMBODIMENTS

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. All UnitedStates patents and published or unpublished United States patentapplications cited herein are incorporated by reference. All publishedforeign patents and patent applications cited herein are herebyincorporated by reference. Genbank and NCBI submissions indicated byaccession number cited herein are hereby incorporated by reference. Allother published references, documents, manuscripts and scientificliterature cited herein are hereby incorporated by reference.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is:
 1. An isolated soluble fusion protein complexcomprising at least two soluble proteins, wherein: a first solubleprotein comprises a polypeptide according to SEQ ID NO:4, and a secondsoluble protein comprises a polypeptide according to SEQ ID NO:2,wherein the first and second soluble proteins are covalently linked by adisulfide bond to form the soluble fusion protein complex.
 2. Thesoluble fusion protein complex of claim 1, wherein the first solubleprotein consists of SEQ ID NO:4 and the second soluble protein consistsof SEQ ID NO:2.
 3. The soluble fusion protein complex of claim 1,wherein the first and/or second soluble protein are associated with abiologically active moiety.
 4. The soluble fusion protein complex ofclaim 3, wherein the biologically active moiety comprises cytokines,antibodies, T cell receptors, receptor binding molecules, receptordomains, immune checkpoint agonists, immune checkpoint antagonists,chemokines, growth factors, toxins, cytotoxic agents, or combinationsthereof.
 5. The soluble fusion protein complex of claim 4, wherein thebiologically active moiety comprises one or more cytokines.
 6. Thesoluble fusion protein complex of claim 1, wherein the first solubleprotein contains an Fc domain and is covalently linked to the secondsoluble protein by a disulfide bond linking the Fc domain of the firstsoluble protein to the Fc domain of the second soluble protein.